N-oxides of amino containing pyrido 2,3-D! pyrimidines

ABSTRACT

N-oxides of amino containing 6-aryl pyrido[2,3-d]-pyrimidine 7-imines, 7-ones, and 7-thiones have the formula where A and B are linkers, Ar is aryl, R2 is aklyl, X is O, S, or NH, or NAcyl, and R5 and R6 are alkyl. The compounds are inhibitors of protein tyrosine kinases and cyclin-dependent kinases, and are thus useful in treating cellular proliferation mediated thereby. The compounds are especially useful in treating cancer, atherosclerosis, restenosis, and psoriasis.

This application claims the benefit of U.S. Provisional ApplicationNumber 60/038,822 filed Feb. 5, 1997.

FIELD OF THE INVENTION

This invention relates to N-oxides of certain pyrido 2,3-d!pyrimidinesand their use in inhibiting cellular proliferation and protein tyrosinekinase enzymatic activity.

BACKGROUND OF THE INVENTION

Numerous pyrido 2,3-d!pyrimidines are known. For example, U.S. Pat. No.3,534,039 discloses a series of 2,7-diamino-6-arylpyrido2,3-d!pyrimidine compounds as diuretic agents; U.S. Pat. No. 3,639,401discloses a series of 6-aryl-2,7-bis (trialkylsilyl)amino!-pyrido2,3-d!pyrimidine compounds as diuretic agents; U.S. Pat. No. 4,271,164discloses a series of 6-substituted-arylpyrido 2,3-d!pyrimidin-7-aminesand derivatives as antihypertensive agents; European PublishedApplication 0 537 463 A2 discloses a series of substituted-pyrido2,3-d!pyrimidines useful as herbicides. More recently, WO96/34867teaches a group of 2-amino-pyrido 2,3-d!pyrimidines which are useful fortreating atherosclerosis, restenosis, psoriasis, and cancer. Thisinvention provides N-oxides of certain tertiary amino containing2-amino-pyrido 2,3-d!pyrimidines. The N-oxides are metabolites of thecorresponding tertiary. amino compounds that also inhibit kinaseenzymes.

SUMMARY OF THE INVENTION

This invention provides new compounds characterized as N-oxides ofcertain 2-amino substituted pyrido 2,3-d!pyrimidines which are useful ininhibiting protein tyrosine kinases and cell cycle dependent kinases,and thus are effective in treating cellular proliferative diseases ofatherosclerosis, restenosis, psoriasis, and cancer. The invention ismore particularly directed to compounds defined by the Formula I##STR2## wherein A and B are linkers which independently are alkylene,alkylenoxy, alkylenethio, or arylene;

X is NH, N-Acyl, O, or S;

R₂ is hydrogen, (CH₂)_(n) Ph, where Ph is phenyl or substituted phenyland n is 0, 1, 2, or 3; heteroaromatic, cycloalkyl, C₁ -C₆ alkanoyl, C₁-C₆ alkyl, C₂ -C₆ alkenyl, and C₂ -C₆ alkynyl, where the alkyl, alkenyl,and alkynyl groups may be substituted by NR₅ R₆, phenyl, substitutedphenyl, thioalkyl, alkyloxy, hydroxy, carboxy, halogen, cycloalkyl, andwhere R₅ and R₆ are independently hydrogen, C₁ -C₆ alkyl, C₂ -C₆alkenyl, C₂ -C₆ alkynyl, (CH₂)_(n) Ph where Ph is phenyl or substitutedphenyl and n is 0, 1, 2, or 3; cycloalkyl, heteroaromatic, and R₅ and R₆taken together with the nitrogen to which they are attached can completea ring having 3 to 7 carbon atoms and optionally containing 1, 2, or 3heteroatoms selected from nitrogen, substituted nitrogen, oxygen, andsulfur;

Ar is phenyl, substituted phenyl, or heteroaromatic;

and the pharmaceutically acceptable salts thereof.

Preferred compounds have the above formula wherein Ar is phenyl orphenyl substituted with 1 or 2 groups selected from C₁ -C₆ alkyl, C₁ -C₆alkoxy, and halo, especially halo such as chloro or bromo.

Further preferred compounds are those wherein R₂ is C₁ -C₆ alkyl, C₂ -C₆alkenyl, (CH₂)_(n) Ph such as phenyl and benzyl, or C₃ -C₆ cycloalkylsuch as cyclopropyl.

An especially preferred group of compounds have the above formulawherein X is O.

Another preferred group of compounds are those wherein X is NH. Theseare imines, and are especially useful as intermediates leading tocompounds where X is O.

Further preferred compounds have the above formula wherein A is C₁ -C₆alkylenoxy and B is phenylene.

An especially preferred group of invention compounds have the formula##STR3## wherein R₂ is C₁ -C₆ alkyl, and R₇ and R₈ independently arehydrogen, C₁ -C₆ alkyl, C₁ -C₆ alkoxy, or halo, especially chloro,fluoro, or bromo; R₅ and R₆ are independently C₁ -C₆ alkyl, or togetherwith the nitrogen to which they are attached complete a cyclic ringhaving 2 heteroatoms, for example ##STR4## where R₉ is hydrogen, C₁ -C₆alkyl, or (CH₂)_(n) Ph.

Another preferred group of compounds have the formula ##STR5## where R₂,R₅, R₆, R₇, and R₈ are as defined above and A is alkylene or alkylenoxyand B is phenylene.

Another preferred group of compounds have the formula ##STR6## where R₂,R₅, R₆, R₇, and R₈ are as defined above.

Another preferred group of compounds have the formula ##STR7##

The compounds from this group that are especially preferred have theformula ##STR8##

Particularly preferred are compounds of the formula ##STR9##

It should be appreciated that when R₂ is hydrogen, the compounds canexist in tautomeric form as follows ##STR10##

The most preferred compounds of the invention have the formula ##STR11##where R₂, R₅, R₆, R₇, and R₈ are as defined above, A is --CH₂ CH₂ --O--,and B is phenylene. Ideally, R₂ is alkyl such as methyl or ethyl, and R₇and R₈ are hydrogen or alkoxy such as methoxy or ethoxy. The mostpreferred Ar group is phenyl, ideally phenyl substituted with one, two,or three groups selected from halo, C₁ -C₆ alkyl, hydroxy, C₁ -C₆alkoxy, carboxy, C₁ -C₆ alkoxycarbonyl, and C₁ -C₆ alkyl substitutedwith hydroxy, carboxy, alkoxycarbonyl, amino, C₁ -C₆ alkylamino, anddi-C₁ -C₆ alkylamino. An especially preferred group of such compoundshave the formula ##STR12##

This invention also provides pharmaceutical formulations comprising acompound of Formula I together with a pharmaceutically acceptablecarrier, diluent, or excipient therefor.

Compounds within the scope of the present invention have a specificaffinity towards one or more of the substrate sites of the tyrosinekinase domains of EGF, and other EGF family of receptors such as erb B2,3, and 4; FGFs, PDGF, V-src, and C-src, as well as the cell cycledependent kinases, such as cdk2, cdk4, cdc2, and wee-1-kinase. Compoundswithin the scope of the present invention have effectively inhibitedPDGF autophosphorylation of the receptor and inhibited vascular smoothmuscle cell proliferation and migration.

As inhibitors of protein kinases and cell cycle dependent kinases, thecompounds of the instant invention are useful in controllingproliferative disorders including leukemia, cancer, psoriasis, vascularsmooth muscle proliferation associated with atherosclerosis, andpostsurgical vascular stenosis and restenosis in mammals, and are usefulin osteoporosis and as immunosuppressants and antiangiogenic agents.

A further embodiment of this invention is a method of treating subjectssuffering from diseases caused by vascular smooth muscle proliferation.The method entails inhibiting vascular smooth muscle proliferationand/or migration by administering an effective amount of a compound ofFormula I to a subject in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention can exist in unsolvated forms aswell as solvated forms, including hydrated forms. In general, thesolvated forms, including hydrated forms, are equivalent to unsolvatedforms and are intended to be encompassed within the scope of the presentinvention.

In the compounds of Formula I, the term "C₁ -C₆ alkyl" means a straightor branched hydrocarbon radical having from 1 to 6 carbon atoms andincludes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, and the like.

"Halo" means fluoro, chloro, bromo, and iodo.

"C₂ -C₆ Alkenyl" means straight and branched hydrocarbon radicals havingfrom 2 to 6 carbon atoms and 1 double bond and includes ethenyl,3-buten-1-yl, 2-ethenylbutyl, 3-hexen-1-yl, and the like. Typical C₂ -C₆alkynyl groups include propynyl, 2-butyn-1-yl, 3-pentyn-1-yl, and thelike.

"C₃ -C₆ Cycloalkyl" means a cyclic hydrocarbyl group such ascyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.

"C₁ -C₆ Alkoxy" refers to the alkyl groups mentioned above bindedthrough oxygen, examples of which include methoxy, ethoxy, isopropoxy,tert-butoxy, and the like.

"C₁ -C₆ Alkanoyl" groups are alkyl linked through a carbonyl, i.e., C₁-C₅ alkyl ##STR13## Such groups include formyl, acetyl, propionyl,butyryl, and isobutyryl.

"Acyl" means an alkyl or aryl (Ar) group bonded through a carbonyl group##STR14## For example, acyl includes a C₁ -C₆ alkanoyl, includingsubstituted alkanoyl, wherein the alkyl portion can be substituted byNR₅ R₆ or a carboxylic or heterocyclic group. Typical acyl groupsinclude acetyl, benzoyl, and the like.

The term "alkylene" means a linker which is a straight or branchedcarbon chain having 1 to 6 carbon atoms and having two bonding sites.Examples of C₁ -C₆ alkylene linker groups include --CH₂ --, --CH₂ CH₂--, ##STR15## and n-pentylmethylene.

The term "alkylenoxy" means a C₁ -C₆ alkylene linker group having aterminal and bonded through an oxygen atom. Examples include ##STR16##

The term "alkylenthio" means a C₁ -C₆ alkylene linker group having aterminal end bonded through a sulfur atom. Examples include --CH₂ --S--,--CH₂ CH₂ --S--, --CH₂ CH₂ CH₂ --S--, and ##STR17##

The alkyl, alkenyl, alkoxy, and alkynyl groups described above may besubstituted. The substituent groups which may be part of the alkyl,alkenyl, alkoxy, and alkynyl groups are NR₅ R₆, phenyl, substitutedphenyl, thioalkyl (C₁ -C₆), C₁ -C₆ alkoxy, hydroxy, carboxy, C₁ -C₆alkoxycarbonyl, halo, cycloalkyl, and a 5- or 6-membered carbocyclicring or heterocyclic ring having 1 or 2 heteroatoms selected fromnitrogen, substituted nitrogen, oxygen, and sulfur. "Substitutednitrogen" means nitrogen bearing C₁ -C₆ alkyl or (CH₂)_(n) Ph.

Examples of substituted alkyl groups thus include 2-aminoethyl,2-diethylaminoethyl, 2-dimethylaminopropyl, ethoxycarbonylmethyl,3-phenylbutyl, methylsulfanylmethyl, methoxymethyl, 3-hydroxypentyl,2-carboxybutyl, 4-chlorobutyl, 3-cyclopropylpropyl, 3-morpholinopropyl,piperazinylmethyl, and 2-(4-methylpiperazinyl) ethyl.

Examples of substituted alkenyl groups thus include2-diethylaminoethenyl, 3-amino-2-butenyl, 3-(1-piperazinyl)-1-propenyl,3-hydroxy-1-propenyl, 2-(1-s-triazinyl)ethenyl, 3-phenyl-3-pentenyl, andthe like.

Examples of substituted alkynyl groups include 2-methoxyethynyl,2-ethylsulfanyethynyl, 4-(1-piperazinyl)-3-(butyryl),3-phenyl-5-hexynyl, 3-diethylamino-3-butyryl, 4-chloro-3-butyryl,4-cyclobutyl-4-hexynyl, and the like.

Typical substituted alkoxy groups include aminomethoxy,trifluoromethoxy, 2-diethylaminoethoxy, 2-ethoxycarbonylethoxy,3-hydroxypropoxy, 6-carboxyhexyloxy, and the like.

Further, examples of substituted alkyl, alkenyl, and alkynyl groupsinclude dimethylaminomethyl, carboxymethyl, 4-diethylamino-3-buten-1-yl,5-ethylmethylamino-3-pentyn-1-yl, 4-morpholinobutyl,4-tetrahydropyridinylbutyl-3-imidazolidin-1-ylpropyl,4-tetrahydrothiazol-3-yl-butyl, phenylmethyl, 3-chlorophenylmethyl, andthe like.

The term "Ar" refers to unsubstituted and substituted aromatic andheteroaromatic groups. Heteroaromatic groups have from 4 to 9 ringatoms, from one to four of which are selected from O, S, and N.Preferred groups have 1 or 2 heteroatoms in a 5- or 6-membered aromaticring. Mono and bicyclic ring systems are included. Typical Ar groupsinclude phenyl, 3-chlorophenyl, 2,6-dibromophenyl, pyridyl,3-methylpyridyl, benzothienyl, 2,4,6-tribromophenyl,4-ethylbenzothienyl, furanyl, 3,4-diethylfuranyl, naphthyl,4,7-dichloronaphthyl, and the like.

Preferred Ar groups are phenyl and phenyl substituted by 1, 2, or 3groups independently selected from halo, alkyl, alkoxy, thio, thioalkyl,hydroxy, alkanoyl, --CN, --NO₂, --COOR₈, --CF₃, alkanoyloxy, or amino ofthe formula --NR₅ R₆. The alkyl and alkoxy groups can be substituted asdefined above. For example, typical groups are ##STR18## preferred, and2,6-disubstituted phenyl is especially preferred.

Typical Ar substituted phenyl groups which are preferred thus include2-aminophenyl, 3-chloro-4-methoxyphenyl, 2,6-dimethylphenyl,2,6-diethylphenyl, 2-n-hexyl-3-fluorophenyl, 3-hydroxyphenyl,4-hydroxymethylphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl,2,6-dichlorophenyl, 4-(3-aminopropoxy)phenyl, 2,6-difluorophenyl,2-chloro-6-methylphenyl, 2,4,6-trichlorophenyl, 2,6-dimethoxyphenyl,4-(diethylaminoethoxy)phenyl, 2,6-dihydroxyphenyl, 2,6-dibromophenyl,2,6-dinitrophenyl, 2,6-di-(trifluoromethyl)phenyl,3-(diethylaminoethyl)phenyl, 3,5-dimethylsulfanylphenyl,3,5-diethylsulfanylphenyl, 2,6-dimethylphenyl, 2,3,6-trimethylphenyl,2,6-dibromo-4-methylphenyl, and 3,5-di-(N,N-dimethylamino)phenyl.

The term "arylene" means a linker defined as Ar having two bonding sites(e.g., --Ar--). Examples include ##STR19## Preferred arylene linkers arephenylene and substituted phenylene.

The compounds of Formula I are capable of further forming bothpharmaceutically acceptable acid addition and/or base salts. All ofthese forms are within the scope of the present invention.

Pharmaceutically acceptable acid addition salts of the compounds ofFormula I include salts derived from inorganic acids such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic,phosphorous, and the like, as well as the salts derived from organicacids, such as aliphatic mono- and dicarboxylic acids,phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioicacids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Suchsalts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, caprylate, isobutyrate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Also contemplated aresalts of amino acids such as arginate and the like and gluconate,galacturonate (see, for example, Berge S. M., et al., "PharmaceuticalSalts," J. of Pharmaceutical Science, 66:1-19 (1977)).

The acid addition salts of the basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge S. M., et al., "Pharmaceutical Salts," J. of PharmaceuticalScience, 66:1-19 (1977)).

The base addition salts of acidic compounds (for example when R₃ is acarboxy alkyl group such as carboxymethyl or 3-carboxybutyl) areprepared by contacting the free acid form with a sufficient amount ofthe desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention.

While the forms of the invention herein constitute presently preferredembodiments, many others are possible. It is not intended herein tomention all of the possible equivalent forms or ramifications of theinvention. It is understood that the terms used herein are merelydescriptive rather than limiting, and that various changes may be madewithout departing from the spirit or scope of the invention.

The N-oxides provided by this invention are readily prepared byoxidizing the corresponding tertiary amine according to the followingscheme. ##STR20##

The oxidation is readily accomplished by reacting the tertiary aminewith an equimolar quantity or excess of a common oxidizing agent, suchas hydrogen peroxide, a peracid such as peracetic acid, orm-chloroperbenzoic acid, or any of the oxaziridine oxidants, such as2-phenylsulforyl-3-phenyloxaziridine and2-phenylsulfonyl-3-(4-nitrophenyl)-oxaziridine. The oxidation of thetertiary amine generally is carried out in an unreactive organic solventsuch as chloroform, carbontetrachloride, tetrahydrofuran,dichloromethane, or acetone, and generally is complete within about 30minutes to about 3 hours when conducted at a temperature of about 0° C.to about 50° C. The N-oxide product can be isolated by simply removingthe reaction solvent, and the product can be further purified, ifdesired, by standard methods such as chromatography, and crystallizationfrom solvents such as isopropyl alcohol or ethanol.

The N-oxides of the invention are metabolites of the correspondingtertiary amines, and accordingly can be obtained by administering atertiary amine to an animal such as a mouse or dog, and isolating thecorresponding N-oxide from the blood plasma or urine.

The tertiary amines, which are starting materials for the N-oxides ofthe invention, may be prepared according to the syntheses outlined inSchemes I-VI. Although these schemes often indicate exact structures,the methods apply widely to analogous compounds, given appropriateconsideration to protection and deprotection of reactive functionalgroups by methods standard to the art of organic chemistry. For example,hydroxy groups, in order to prevent unwanted side reactions, generallyneed to be converted to ethers or esters during chemical reactions atother sites in the molecule. The hydroxy protecting group is readilyremoved to provide the free hydroxy group. Amino groups and carboxylicacid groups are similarly derivatized to protect them against unwantedside reactions. Typical protecting groups, and methods for attaching andcleaving them, are described fully by Greene and Wuts in ProtectiveGroups in Organic Svnthesis, John Wiley and Sons, New York, (2nd Ed;1991), and McOmie, Protective Groups in Organic Chemistry, Plenum Press,New York, 1973.

Scheme I describes a typical method for preparing the pyrido2,3-d!pyrimidin-7(8H)-ones and the 7-(8H)-imides, compounds wherein X isO or NH. The synthesis starts by reacting a cyanoacetate such as ethylethoxymethylenecyanoacetate with a thiopseudourea such as2-methyl-2-thiopseudourea sulfate to provide5-cyano-4-hydroxy-2-(methylsulfanyl)pyrimidine. This reaction isdescribed more fully in Helv. Chim. Acta., 42:763-772 (1959). The4-hydroxypyrimidine is next reacted with a halogenating agent such asphosphorous oxychloride or thionyl chloride to provide a 4-halopyrimidine, for example, 5-cyano-4-chloro-2-(methylsulfanyl)pyrimidine.The halopyrimidine next is reacted with an amine R₂ NH₂ to provide a5-cyano-4-substituted amino-2-(methylsulfanyl)pyrimidine. The amineutilized can have R₂ be the group desired in the final product, forexample, alkyl such as methyl, or R₂ can be a group that can be laterremoved, for example, benzyl or the like, to generate compounds whereinR₂ is hydrogen. Compounds where R₂ is hydrogen can be alkylated andacylated by standard methods.

The reaction between the halopyrimidine and the amine R₂ NH₂ typicallyis carried out by mixing equimolar quantities of the halopyrimidine andamine in an unreactive organic solvent such as toluene, xylene,methylene chloride, or the like, at a temperature of about 50° C. toabout 150° C. Excess amine can be utilized if desired. The4-aminopyrimidine that is produced is next reacted with hydrazine or asubstituted hydrazine to displace the 2-methylsulfanyl group to providea 2-hydrazino-4-substituted amino-5-cyano-pyrimidine. Thehydrazino-pyrimidine is reacted with sodium nitrite in aqueous mineralacid to effect diazotization of the hydrazine group to provide a2-azido-4-(substituted amino)-5-cyano-pyrimidine. Reaction of thiscompound with a reducing agent such as Raney Nickel effectshydrogenation of both the cyano group and the azido group to produce a2-amino-4-(substituted amino)-5-pyrimidinecarboxaldehyde.

The 4-(substituted amino)-5-pyrimidine carboxaldehydes can alternativelybe prepared by starting with a commercially available4-halo-5-pyrimidinecarboxylic acid ester. For example,2-methylsulfanyl-4-chloro-5-pyrimidinecarboxylic acid ethyl ester(available from Aldrich Co.) can be reacted with an amine R₂ NH₂, suchas methylamine, benzylamine, or the like, to displace the 4-chloro groupand provide the corresponding 2-methylsulfanyl-4-(substitutedamino)-5-pyrimidinecarboxylic acid ethyl ester. The in ester group isreduced to an alcohol, for instance by reaction with lithium aluminumhydride in tetrahydrofuran, and the alcohol group is then oxidized to analdehyde by reaction with an oxidant such as sodium dichromate,manganese II oxide, or the like, to give the corresponding2-methylsulfanyl-4-(substituted amino)-5-pyrimidinecarboxaldehyde. The2-methyl sulfanyl group is displaced with hydrazine, and the hydrazinogroup is diazotized and subsequently reduced as described above toprovide the desired 2-amino-4-(substitutedamino)-5-pyrimidinecarboxaldehyde.

The pyrimidinecarboxaldehyde is next reacted with an arylacetonitrile inthe presence of a base and in a solvent such as xylene, 2-ethoxyethanol,dioxane, or the like, as shown in Scheme I. Typical bases that can beutilized include sodium hydride, sodium methoxide, sodium metal,potassium carbonate, and the like. The pyrimidine carboxaldehyde andarylacetonitrile are typically utilized in approximately equimolarquantities. Typical arylacetonitriles which can be employed includephenylacetonitrile, 2,6-dichlorophenylacetonitrile,2,6-dimethylphenylacetonitrile, o-tolylacetontrile, pyridylacetonitrile,furanylacetonitrile, naphthylacetonitrile, and the like. The reactiontypically is carried out in an unreactive solvent such as methyl orethyl cellosolve, diglyme, dimethylformamide, or the like, and at anelevated temperature of about 50° C. to about 200° C., and generally issubstantially complete within about 2 to about 24 hours. The product, a6-aryl-7-imino-8-substituted-7,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine, wherein X is NH, and R₁ is NR₃ R₄, is readilyisolated by adding water to the reaction mixture, which generally causesprecipitation of the product. The product imine can be further purifiedif needed by recrystallization from solvents such as ethyl acetate,acetone, isopropanol, and the like, or by chromatography over solidsupports such as silica gel. Heating the 7-imino compound in thepresence of an acid such as hydrochloric acid produces the corresponding7-oxo compound.

The 6-aryl-7-imino(and 7-oxo)-8-substituted-7,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine thus prepared has the formula ##STR21##wherein R₂ and Ar are as defined above, R₃ is hydrogen, and R₄ ishydrogen or the group NR₅ R₆ --(A)₀ or 1 -(B)₀ or 1 -, where A, B, R₅,and R₆ are as defined above. Typical imines and ketones thus preparedinclude the following:

    __________________________________________________________________________    R.sub.2                                                                              R.sub.3                                                                         R.sub.4            Ar                                                __________________________________________________________________________    CH.sub.3                                                                             H CH.sub.2 N(Et).sub.2                                                                             phenyl                                            cyclopropyl                                                                          H CH.sub.2 N(Me).sub.2                                                                             3-methoxyphenyl                                   3-butyryl                                                                            H CH.sub.2 --O--CH.sub.2 N(Me).sub.2                                                               1-naphthyl                                        3-chlorophenyl                                                                       H                                                                               1 #STR22##         3-pyridyl                                         3-aminopropyl                                                                        H                                                                               2 #STR23##         2-thienyl                                         benzyl H                                                                               3 #STR24##         2,3,5-tribromophenyl.HCl                          Et     H                                                                               4 #STR25##         phenyl                                            Et     H                                                                               5 #STR26##         phenyl                                            Et     H                                                                               6 #STR27##         2-iodophenyl                                      Me     H                                                                               7 #STR28##         2,6-dibromophenyl                                 Me     H                                                                               8 #STR29##         3,5-dimethoxyphenyl                               iPr    H                                                                               9 #STR30##         3,4-dimethylsulfonylphenyl                        __________________________________________________________________________

The 6-aryl-7-imino-8-substituted-7,8-dihydro-pyrido2,3-d!pyrimidine-2-ylamines are useful therapeutic agents, as well asintermediates since they can be oxidized to the corresponding N-oxidesof the invention, and additionally they are readily converted to thecorresponding 7-keto derivative by simply heating in a mineral acid suchas hydrochloric acid, sulfuric acid, phosphoric acid, or the like. Thehydrolysis generally is substantially complete after about 5 to about 24hours when carried out at about 60° C. to about 200° C. The product, a2-amino-6-aryl-8-substituted-pyrido 2,3-d!pyrimidin-7(8H)-one, isreadily isolated by removal of the reaction solvent, for example byevaporation under reduced pressure, and crystallization from commonsolvents such as ethyl acetate, acetone, tetrahydrofuran, and the like.

The 7-oxo-pyrido 2,3-d!pyrimidines can alternatively be prepared bysimply hydrolyzing a 7-amino-pyridopyrimidine in a mineral acid, asillustrated in Scheme II. The 7-amino-pyridopyrimidines are readilyavailable by the methods described in U.S. Pat. No. 3,534,039. The7-amino-pyridopyrimidine is simply dissolved in a mineral acid such asconcentrated hydrochloric acid, sulfuric acid, phosphoric acid, or thelike. The hydrolysis reaction generally is complete after about 12 toabout 24 hours when carried out at about 80° C. to about 200° C. Theproduct is readily isolated by removal of the reaction solvent andcrystallization from a solvent such as dimethylsulfoxide,dimethylformamide, dioxane, or the like. The primary 2-amino(NH₂ -)derivatives can then be alkylated and oxidized to give the N-oxides ofthis invention.

The 7-oxo-pyrido 2,3-d!pyrimidines can alternatively be prepared byreacting a 2,4-diamino-5-pyrimidinecarboxaldehyde with an arylacetoester as shown below: ##STR31## where R₂, A, B, R₅, R₆, and Ar areas defined above, and alkyl is a lower alkyl group such as methyl,ethyl, isobutyl, and the like. The reactants generally are mixedtogether in an unreactive solvent such as dimethylformamide,tetrahydrofuran, or ethyl cellosolve, and the aryl acetoester generallyis utilized in excess, for instance in a 0.5 to 1.0 molar excessrelative to the pyrimidine. The reaction is carried out in the presenceof a base such as sodium methoxide or sodium hydride, and generally iscomplete within about 2 to about 24 hours when carried out at anelevated temperature of about 50° C. to about 120° C. The product7-oxo-pyrido 2,3-d!pyrimidines are recovered by removing the reactionsolvents and crystallizing the product from an organic solvent such asmethanol, ethyl acetate, or the like.

Invention compounds wherein R₂ in Formula I is other than hydrogen arereadily prepared by utilizing a substituted amine R₂ NH₂ in the reactiondescribed above, or alternatively by alkylating a pyridopyrimidinewherein R₂ is hydrogen, for example as illustrated in Scheme II. Thereaction generally is carried out by mixing the pyridopyrimidine with anequimolar quantity or excess of alkylating agent, for instance an alkylhalide such as methyl iodide, benzyl bromide, 3-hexen-1-yl iodide, orthe like, in a mutual isolvent such as toluene, xylene,dimethylformamide, or the like. A base such as sodium hydride can beadded to catalyze the reaction and to act as an acid scavenger. Theproduct, an 8-substituted pyridopyrimidine, is readily isolated byremoval of the reaction solvents, and further purified if desired bychromatography or crystallization from toluene, acetone, or the like.

Scheme III illustrates the reaction of 2-amino-pyridopyrimidines withacylating agents and diacylating agents to form amides and cyclic aminosystems. For example, a 2-amino-pyridopyrimidine of the formula##STR32## wherein R₂ is other than hydrogen, X is O or S, and Ar is asdefined above, can be reacted with an equimolar quantity or slightexcess of an acid halide or an acid anhydride to effect acylation of the2-amino group. Typical acid halides include acetyl chloride, benzoylbromide, propionyl iodide, and the like. Commonly used a anhydridesinclude acetic anhydride, propionyl anhydride, and mixed anhydrides suchas acetic butyric anhydride. Acylating agents such as succinic anhydrideand the like can be utilized to form cyclic imides as described inScheme III.

Invention compounds wherein X is S have the formula ##STR33## whereinR₁, R₂, and Ar are as defined above. These pyridopyrimidine thiones areprepared by reacting the corresponding 7-oxo compounds (i.e., where X=O)with an equivalent amount of Lawesson's Reagent or phosphoruspentasulfide in a solvent, preferably pyridine or toluene, at anelevated temperature of about 90° C. to about 125° C. for a period ofabout 1 to about 24 hours. The product is readily isolated by simplyremoving all reaction solvent, and further purification can be achieved,if desired, by routine methods such as crystallization, chromatography,and the like.

The 2-amino- and 2-alkylsulfanyl-pyridopyrimidines, compounds of theformula ##STR34## can alternatively be prepared as described in SchemeIV.

Scheme IV describes a method of synthesis of compounds having a basicside chain at the 2-position of the pyrido 2,3-d!pyrimidine ring system,for example, where R₁ is NR₃ R₄, R₃ is hydrogen, and R₄ is C₁ -C₆ alkylsubstituted with NR₅ R₆. In the first step, an aldehyde such as4-methylamino-2-methylsulfanyl-pyrimidine-5-carbaldehyde is condensedwith a arylacetonitrile derivative such as2,6-dichlorophenyl-acetonitrile, in a mutual solvent such asN,N-dimethylformamide, and in the presence of a 1 to 5 molar excess of abase, preferably powdered potassium carbonate or cesium carbonate, attemperatures preferably in the range of 110° C. to 153° C. for a timeperiod of 0.5 to 25 hours. The resulting 7-imino-2-methylsulfanylderivatives are useful for preparing a variety of 2-amino derivatives.For example, treatment with a 100 to 500 percent molar excess of aprimary amine such as N,N-diethylaminopropylamine, at temperatures inthe range of 100° C. to 150° C. for about 1 to about 24 hours, gives thecorresponding 2-substituted amino derivatives. In the case of aminesthat boil at less than about 100° C., e.g., methylamine, ethylamine,propylamine, and the like, an appropriate pressure bomb can be utilizedto reach the desired reaction temperatures. The resulting2-amino-7-imino derivatives are readily hydrolyzed, if desired, to the2-amino-7-oxo derivatives by reaction with a strong mineral acid, suchas concentrated hydrochloric acid or sulfuric acid, at refluxtemperatures for prolonged periods of time, in the range of 6 hours to 7days.

Alternatively, the 7-imino-2-methylsulfanyl derivatives can be acylatedby reaction with an acyl halide or acyl anhydride, for example acetylchloride or propionic anhydride, to provide the corresponding7-acylimino-2-methylsulfanylpyridopyrimidines. These compounds can bereacted with an amine as described above to effect displacement of the2-methylsulfanyl group and provide a 2-aminopyridopyrimidine having anacylimino group at the 7-position (i.e., X=N Acyl). The 7-acylimidoderivatives can be reacted with a strong acid as described above toeffect hydrolysis of the 7-acylimino group to a 7-oxo group.

The sulfanyl compounds of Formula I, i.e., where R₁ is SR₃, are readilyoxidized to the corresponding sulfoxides and sulfones by reaction withagents such as m-chloroperbenzoic acid, hydrogen peroxide, sodiumperborate, potassium hydrogen persulfate, and the like. As noted above,the sulfanyl, sulfinyl, and sulfonyl derivatives of Formula I areespecially useful to make the corresponding amino derivatives, becausethey readily react with amines (HNR₃ R₄) to undergo nucleophilicdisplacement. This is an especially preferred method for making the2-arylamino and 2-heteroarylamino compounds of the invention (e.g.,where R₃ is phenyl or pyridyl). The foregoing oxidation and nucleophilicdisplacement reactions are illustrated in Scheme V.

The oxidation of a sulfanyl compound of Formula I is accomplished byreacting it with an equimolar quantity of an oxidant, preferablym-chloroperbenzoic acid, to produce the corresponding sulfoxide, or witha two molar equivalent to produce the corresponding sulfone. Theoxidation typically is carried out in an organic solvent such aschloroform or dichloromethane, and typically is complete within 1 to 24hours when carried out at 25° C. to 45° C. Larger quantities of oxidantand longer reaction times ensure complete formation of the sulfone. Thecorresponding sulfoxide or sulfone is readily isolated by filtration, orby removal of the reaction solvent by evaporation under reducedpressure.

Amines readily displace the sulfanyl, sulfinyl, and sulfonyl groups toproduce compounds of Formula I where R₁ is NR₃ R₄. This is an especiallypreferred method for making aryl amino compounds, i.e., phenylamino,substituted phenylamino, heteroarylamino (e.g., pyridylamino,thienylamino), and substituted heteroarylamino (e.g.,ethylpyridylamino). The displacement is accomplished by mixing thesulfanyl, sulfinyl, or sulfonyl compound with an amine, preferably aprimary or secondary amine. The amine generally is utilized in excess,for instance from about 20 to 500 molar excess relative to the sulfanyl,sulfinyl, or sulfonyl compound. The reactants generally are mixed neator in a mutual organic solvent, for example, dimethylformamide,(ethoxyethyl)ether, glacial acetic acid, dimethylsulfoxide, and thelike. The reaction generally is complete after about 5 minutes to about6 hours when carried out at an elevated temperature of about 100° C. toabout 250° C. The product, a compound of Formula I, where R₁ is NR₃ R₄,is readily isolated by filtration, or by removing the reaction solventby evaporation. The product can be purified further, if desired, bycrystallization, chromatography, or the like.

The 2-amino pyridopyrimidines of the invention (Formula I where R₁ isNR₃ R₄) can alternatively be prepared by reacting a2,4-diamino-5-pyrimidinecarboxaldehyde with an aryl acetonitrile (ArCH₂CN). The 2,4-diamino-5-pyrimidinecarboxaldehydes can be prepared fromreadily available 2-sulfanyl or sulfinyl pyrimidines by nucleophilicdisplacement with an amine as described above. For example, a2-sulfanyl-4-substituted amino-5-alkoxycarbonylpyrimidine can be reactedwith an oxidizing agent such as oxaziridine to give the correspondingsulfoxide or sulfone. The sulfoxide or sulfone substituent is readilydisplaced by reaction with an amine (HNR₃ R₄), to provide thecorresponding 2,4-diamino-5-alkoxycarbonylpyrimidine. The alkoxycarbonylmoiety can be converted to an aldehyde moiety by standard methods (i.e.,reduction to an alcohol and oxidation of the alcohol to an aldehyde).The 2,4-diamino-5-pyrimidinecarboxaldehyde readily reacts with anarylacetonitrile to produce a 2-amino-6-aryl-pyridopyrimidine of theinvention. The foregoing reactions are depicted in Scheme VII.

As noted above, some of the compounds of the invention are basic innature, by virtue of a substituent group which is basic, such as aminogroups for example. Compounds of Formula I wherein R₁ is R₁ NR₃ R₄ aretypically basic. Such basic compounds readily form pharmaceuticallyacceptable salts with any number of inorganic and organic acids. Thesalts typically are crystalline, and generally are water soluble and arethus well suited to oral administration and the like. ##STR35## where Lis a leaving group such as chloro or bromo, and m is an integer of 1, 2,3, or 4. ##STR36##

The following detailed examples further illustrate synthesis of thecompounds of this invention. The examples are illustrative only, and arenot to be construed as limiting the invention in any respect.

EXAMPLE 1 5-Cyano-4-hydroxy-2-(methylsulfanyl)-pyrimidine

To a solution of freshly distilled ethyl ethoxymethylene cyanoacetate(118.99 g) in methanol (800 mL) at 5° C. was added2-methyl-2-thiopseudourea (107.69 g). To this mixture was added asolution of sodium methoxide in methanol prepared by dissolving sodiummetal (35.59 g) in methanol (800 mL). The reaction was allowed to warmto room temperature and stirred for 6 hours. After standing overnight,the solvent was removed under reduced pressure, the residue wasdissolved in 1500 mL of water at 50° C. with stirring, and the solutionwas filtered hot. The filtrate was acidified to pH 2 with concentratedHCl and allowed to stand overnight at room temperature. The product wascollected by filtration and dried to give 48.33 g of5-cyano-4-hydroxy-2-methylsulfanyl-pyrimidine. This product was useddirectly in the next step without further purification.

EXAMPLE 2 4-Chloro-5-cvano-2-methylsulfanyl-pyrimidine

A mixture of 5-cyano-4-hydroxy-2-methylsulfanyl-pyrimidine (48.33 g)from Example 1 and phosphorus oxychloride (150 mL) was heated at refluxfor 3 hours. The reaction mixture was allowed to cool to roomtemperature, filtered, and the filtrate was concentrated to drynessunder vacuum. The residue was partitioned between methylene chloride andice water. The organic layer was separated, washed with water, driedover magnesium sulfate, filtered, and evaporated under reduced pressure.The residue was heated to reflux in hexane (750 mL) w ith stirring. Th ehot hexane so lution was decanted from the insoluble material andallowed to cool to room temperature to afford 32 g of the title compound4-chloro-5-cyano-2-methylsulfanyl-pyrimidine.

EXAMPLE 3 5-Cyano-4-methylamino-2-methylsulfanyl-pyrimidine

Through a cold (5° C.) solution of4-chloro-5-cyano-2-methylsulfanyl-pyrimidine from Example 2 in diethylether (700 mL) was bubbled methylamine gas for a period of 20 minutes.The reaction mixture was stirred for 30 minutes at 5° C., then allowedto warm to room temperature and stirred overnight. Thin layerchromatography on silica gel plates indicated the reaction wasincomplete. The reaction mixture was recooled to 5° C. and methylaminegas bubbled through the suspension with stirring for another 20 minutes.The reaction mixture was stirred at 25° C. for 6 hours, then allowed tostand overnight. The insoluble product was collected and suspended inwater with stirring. The suspension was filtered and the product driedin vacuo to afford 25.87 g of the title compound5-cyano-4-methylamino-2-methylsulfanyl-pyrimidine; mp 185-187° C.

Analysis calculated for C₇ H₈ N₄ S: C, 46.65; H, 4.47; N, 31.09; Found:C, 46.62; H, 4.61; N, 31.43.

EXAMPLE 4 5-Cyano-2-hydrazino-4-methylamino-pyrimidine

A mixture of 5-cyano-4-methylamino-2-methylsulfanyl-pyrimidine (25.86 g)from Example 3 and hydrazine hydrate (52 mL) in ethanol (250 mL) washeated at reflux with stirring for 3 hours. The reaction mixture wascooled to room temperature and the insoluble product was collected byfiltration, washed with cold aqueous ethanol (1:1) to give 23 g of thetitle compound. Crystallization from ethanol afforded an analyticallypure sample of 5-cyano-2-hydrazino-4-methylamino-pyrimidine; mp 247-249°C.

Analysis calculated for C₆ H₈ N₆ : C, 43.90; H, 4.91; N, 51.21; Found:C, 44.05; H, 4.92; N, 51.39.

EXAMPLE 5 2-Azido-5-cyano-4-methylamino-pyrimidine

To a cold (5° C.) solution of5-cyano-2-hydrazino-4-methylamino-pyrimidine (21.7 g) from Example 4 ina mixture of water (260 mL) and concentrated HCl (26.5 mL) was addeddropwise a solution of NaNO₂ (10.03 g) in water (25 mL) with overheadmechanical stirring. A white precipitate formed and after the additionwas completed, the reaction was stirred for an additional 20 minutes at5° C. The insoluble product was filtered and washed with cold water togive 22.4 g of the title compound after drying at 23° C. under highvacuum overnight. Crystallization from ethanol provided an analyticallypure sample of 2-azido-5-cyano-4-methylamino-pyrimidine; mp 225-230° C.

Analysis calculated for C₆ H₅ N₇ : C, 41.14; H, 2.88; N, 55.99; Found:C, 40.88; H, 2.81; N, 55.82.

EXAMPLE 6 2-Amino-4-methylamino-5-pyrimidinecarboxaldehyde

To a suspension of 2-azido-5-cyano-4-methylamino-pyrimidine (22.24 g)from Example 5 in 400 mL of 50% aqueous formic acid was added RaneyNickel catalyst (5 g). The reaction mixture was shaken under anatmosphere of hydrogen (40.1 psi) in a Parr hydrogenation apparatus.There was a vigorous evolution of gas as the mixture was shaken at roomtemperature. After 30 minutes the apparatus was vented, additional RaneyNickel (5 g) was added, the apparatus recharged with hydrogen, and themixture shaken overnight. The catalyst was removed by filtration and thefiltrate was evaporated under high vacuum. The residue was suspended inwater and filtered. The insoluble material was collected and dissolvedin 450 mL of boiling water. The aqueous solution was filtered and the pHof the filtrate was adjusted to 7 with 1N sodium hydroxide. Theprecipitated product was collected by filtration and recrystallized fromethanol to give 5.0 g of2-amino-4-methylamino-5-pyrimidinecarboxaldehyde.

EXAMPLE 7 6-(2,6-Dimethylphenyl)-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine

To 2-ethoxyethanol (7 mL) at -10° C. was added cautiously sodium hydride(60% suspended in mineral oil, 83 mg, 2.08 mmol) with stirring. Themixture was allowed to warm to room temperature and2,6-dimethylphenylacetonitrile (1.5 g, 10.33 mmol) was added, followedby 2-amino-4-methylamino-5-pyrimidinecarboxaldehyde (1.5 g, 9.86 mmol)from Example 6. The resulting reaction mixture was heated at reflux for2 hours, allowed to cool to room temperature, and poured into water. Theinsoluble crude product was collected and dried on the filter. Theproduct was purified by dissolving in boiling ethyl acetate and addinghot hexane to the point just before precipitation. The hot solution wasfiltered and upon cooling the product precipitated to give 1.22 g of6-(2,6-dimethylphenyl)-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine; mp 197-198° C.

Analysis calculated for C₁₆ H₁₇ N₅.0.15 H₂ O: C, 68.14; H, 6.18; N,24.83; Found: C, 68.19; H, 6.14; N, 24.60.

EXAMPLE 8 6-(2-Methylihenyl)-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine

The title compound was prepared in similar manner to that describedabove in Example 7 starting from 2-methylphenylacetonitrile (0.72 g,5.45 mmol) and 2-amino-4-methylamino-5-pyrimidinecarboxaldehyde (0.79 g,5.19 mmol). As described above, sodium hydride (60% suspension inmineral oil, 0.083 g, 2.08 mmol), and 2-ethoxyethanol were employed asthe respective base and solvent. The product was purified bycrystallization from ethyl acetate-hexane to give 0.68 g of6-(2-methylphenyl)-7-imino-8-methyl-77,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine; mp 189-190° C.

Analysis calculated for C₁₅ H₁₅ N₅ : C, 67.91; H, 5.70; N, 26.40; Found:C, 67.52; H, 5.71; N, 26.33.

EXAMPLE 9 6-Phenyl-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!-pyrimidin-2-ylamine

The title compound was prepared in a similar manner as described abovein Example 7 starting from phenylacetonitrile (6.5 mL) and2-amino-4-methylamino-5-pyrimidinecarboxaldehyde (8.10 g). However,sodium methoxide (0.5 g) was used in place of sodium hydride in thisreaction. The product was purified by recrystallization from isopropylalcohol to give 9.2 g of 6-phenyl-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!-pyrimidin-2-ylamine; mp 201-203° C.

Analysis calculated for C₁₄ Hl₃ N₅ : C, 66.91; H, 5.21; N, 27.87; Found:C, 66.74; H, 5.22; N, 27.90.

EXAMPLE 10 2,7-Diamino-6-(2,6-dichlorophenyl)-pyrido 2,3-d!-pyrimidine

(Prepared by the method of U.S. Pat. No. 3,534,039). To a solution ofsodium 2-ethoxyethoxide prepared from 0.14 g of sodium and 60 mL of2-ethoxyethanol was added 2.07 g of2,4-diamino-5-pyrimidinecarboxaldehyde, and 2.79 g of2,6-dichlorophenylacetonitrile. The mixture was heated at reflux for 4hours, allowed to cool to room temperature, and the precipitated productwas filtered and washed with diethyl ether to give2,7-diamino-6-(2,6-dichlorophenyl)-pyrido 2,3-d!-pyrimidine; mp 325-332°C.

EXAMPLE 11 2-Amino-6-(2,6-dichlorophenyl)-pyridor 2,3-d!pyrimidin-7-ol

A solution of 2,7-diamino-6-(2,6-dichlorophenyl)-pyrido 2,3-d!pyrimidine(30.6 g) from Example 10 in concentrated HCl (200 mL) was heated atreflux for 24 hours. The reaction mixture was allowed to cool to roomtemperature, filtered, washed with water, and dried in vacuo to give16.5 g of the crude product. The filtrate was refluxed for another 24hours and upon cooling, yielded an additional 8.8 g of product. The twocrops were combined and recrystallized from dimethylformamide, washedtwice with diethyl ether, and dried in vacuo to afford 5.9 g of2-amino-6-(2,6-dichlorophenyl)-pyrido 2,3-d!pyrimidin-7-ol; mp dec 410°C.

Analysis calculated for C₁₃ H₈ Cl₂ N₄ O: C, 50.84; H, 2.62; N, 18.24;Found: C, 50.45; H, 2.87; N, 18.09.

EXAMPLE 12 2-Amino-6-(2,6-dichlorophenyl)-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-one

To a mixture of 2-amino-6-(2,6-dichlorophenyl)-pyrido2,3-d!pyrimidin-7-ol (3.7 g) from Example 11 in dimethylformamide wasadded NaH (50% suspension in mineral oil, 0.64 g). The resulting slurrywas heated at 65° C. for 0.5 hour until a solution formed. It was thencooled to 50° C. and a solution of methyl iodide (2.0 g) indimethylformamide (10 mL) was added dropwise to the reaction. Thereaction mixture was warmed and kept between 60° C.-80° C. for 3 hours.Upon cooling to room temperature, the reaction mixture was poured intoice water. The insoluble white product was filtered, washed with water,and recrystallized from ethanol using charcoal to give 1.9 g of2-amino-6-(2,6-dichlorophenyl)-8-methyl-pyrido2,3-d!pyrimidin-7(8H)-one; mp 235-237° C.

Analysis calculated for C₁₄ H₁₀ C₁₂ N₄ O: C, 52.36; H, 3.14; N, 17.44;Found: C, 52.03; H, 3.24; N, 17.46.

EXAMPLE 13 2-Amino-6-(2,6-dimethylphenyl)-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-one

A mixture of 6-(2,6-dimethylphenyl)-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine (0.96 g) from Example 7 and aqueous 6N HCl (25mL) was heated at reflux for 2 days. The mixture was allowed to cool toroom temperature and stand overnight at ambient temperature. Aninsoluble white solid was collected by filtration, washed with water,and air dried. The crude product was dissolved in hot ethanol, addinghot ethyl acetate to the point just before precipitation, and filteringthe hot solution. Upon cooling, the pure product crystallized to give 25mg of 2-amino-6-(2,6-dimethylphenyl)-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-one; mp gradually dec over 235° C.

Analysis calculated for C₁₆ H₁₆ N₄ O. 1 HCl.0.15 H₂ O: C, 59.38; H,5.53; N, 17.31; Found: C, 59.42; H, 5.37; N, 17.53.

EXAMPLE 14 2-Amino-6-(2-methylphenyl)-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-one

To a mixture of 6-(2-methylphenyl)-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-ylamine (0.30 g) from Example 8 and concentrated HCl(0.6 mL) was added water (11 mL). The reaction mixture was refluxed for20 hours, then allowed to cool to room temperature. The whiteprecipitate from the reaction mixture was filtered and washed withwater. The product was dried in vacuo to give 0.21 g of2-amino-6-(2-methylphenyl)-8-methyl-pyrido 2,3-d!pyrimidin-7(8H)-one; mp239-241° C.

Analysis calculated for C₁₅ H₁₄ N₄ O.1.46 HCl: C, 56.45; H, 4.88; N,17.55; Found: C, 56.47; H, 4.68; N, 17.59.

EXAMPLE 15 N- 6-(2,6-Dichlorophenyl)-7-oxo-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl!-succinamic acid

A mixture of 0.40 g (1.25 mmol) of2-amino-6-(2,6-dichlorophenyl)-8-methyl-pyrido 2,3-d!pyrimidin-7(8H)-one(from Example 12) and 2.00 g (10.0 mmol) of succinic anhydride wasreacted at 145° C. After 10 minutes the homogeneous melt was cooled andtriturated with 25 mL of water. The mixture was heated at the boilingpoint for 5 minutes to hydrolyze excess anhydride. The mixture wasfiltered hot, and the cake was washed with 10 mL of boiling water. Thedried cake (wt 0.50 g) was triturated with 8 mL of methanol: chloroform(1:20). The insoluble solid was filtered and washed with 1 mL of thesame solvent to give 0.037 g of the pure title compound; mp 214-218° C.

Analysis calculated for C₁₈ H₁₄ C₁₂ N₄ O₄. 0.8 H₂ O: C, 49.62; H, 3.61;N, 12.86; Found: C, 49.26; H, 3.16; N, 12.83.

EXAMPLE 16 4-Methylamino-2-methylsulfanyl-pyrimidine-5-carboxaldehyde

A solution of 4.00 g (0.022 mol) of5-cyano-4-methylamino-2-methylsulfanyl-pyrimidine (from Example 3) in150 mL of 50% aqueous formic acid was reacted with 6.0 g of water-wetRaney Nickel. The mixture was stirred at 25° C. for 12 hours. The solidswere filtered and washed with 40 mL of 50% aqueous formic acid. With icebath cooling, a cold saturated solution of potassium carbonate was addedslowly to the green filtrate until complete precipitation of a solid wasachieved (pH is still acidic; pH about 5). The solid was extracted into200 mL of ethyl acetate, and the solution was dried (potassiumcarbonate), filtered, and concentrated; wt 2.30 g (57%); mp 98-100° C.;tlc (1:1 hexane:ethyl acetate) one spot Rf 0.5.

Mass spectrum (CI) 184 (M+1). Analysis calculated for C₇ H₉ N₃ OS: C,45.89; H, 4.95; N, 22.93. Found: C, 46.24; H, 4.88; N, 23.11.

EXAMPLE 16A Alternative synthesis of4-methylamino-2-methylsulfanyl-pyrimidine-5-carboxaldehyde

To a solution of 4-chloro-2-methylsulfanyl-5-pyrimidinecarboxylate ethylester (18.66 g, 80.4 mmol) in 260 mL of tetrahydrofuran was addedtriethylamine (34 mL, 244 mmol), followed by 30 mL of a 40% aqueoussolution of methylamine. The solution was stirred for 30 minutes at 25°C. then concentrated in vacuo and partitioned between chloroform andsaturated aqueous sodium bicarbonate. The organic layer was washed withbrine, dried over MgSO₄, filtered, and concentrated to provide a whitesolid. The solid was suspended in hexane and filtered to provide 14.70 g(81%) of 4-methylamino-2-methylsulfanyl-5-pyrimidinecarboxylate ethylester; mp 91-93° C.

Analysis calculated for C₉ H₁₃ N₃ O₂ S: C, 47.56; H, 5.76; N, 18.49.Found: C, 47.93; H, 5.67; N, 18.58.

A solution of 4-methylamino-2-methylsulfanyl-5-pyrimidinecarboxylateethyl ester (4.36 g, 19.3 mmol) in 60 mL of tetrahydrofuran was addeddropwise to a room temperature suspension of lithium aluminum hydride(1.10 g, 29.0 mmol) in 40 mL of tetrahydrofuran. After 10 minutes thereaction was carefully quenched with 2 mL of water, 2 mL of 15% NaOH,and an additional 7 mL of water. The mixture was stirred for 1 hour, andthe white precipitate which had formed was removed by filtration, andwas washed with ethyl acetate. The filtrate was concentrated in vacuoand 3:1 hexane:ethyl acetate was added. The solids were collected togive 2.99 g (84%) of4-methylamino-2-methylsulfanyl-5-pyrimidinemethanol; mp 155-157° C.

Analysis calculated for C₇ H₁₁ N₃ OS: C, 45.39; H, 5.99; N, 22.68.Found: C, 45.42; H, 5.93; N, 22.42.

4-Methylamino-2-methylsulfanyl-5-pyrimidinemethanol (2.40 g, 13.0 mmol)in 7 mL of acetic acid was added to a solution of sodiumdichromate-dihydrate (1.30 g, 4.4 mmol) in 6 mL of acetic acid. After 2hours at room temperature, additional sodium dichromate-dihydrate (0.3g, 1.0 mmol) in 1 mL of acetic acid was added. After a total reactiontime of 3.5 hours, the bright yellow solid was removed by filtration.Water (30 mL) was added to the filtrate, followed by aqueous ammoniumhydroxide until basic (pH 9.0). The mixture was cooled in therefrigerator for 30 minutes. The precipitate was collected and dissolvedin ethyl acetate, and the solution was dried over MgSO4. Filtration andconcentration in vacuo gave 0.72 g (30%) of4-methylamino-2-methylsulfanyl-5-pyrimidinecarboxaldehyde; mp 99-101° C.

Analysis calculated for C₇ H₉ N₃ OS: C, 45.89; H, 4.95; N, 22.93. Found:C, 45.80; H, 4.96; N, 22.86.

EXAMPLE 17 6-(2,6-Dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!-pyrimidin-7-ylideneamine

Powdered potassium carbonate (0.8 g; 5.8 mmol) was added to a solutionof 0.220 g (1.2 mmol) of the aldehyde from Example 18 and 0.235 g (1.26mmol) (ca. 5% excess) of 2,6-dichlorophenylacetonitrile in 2.0 mL ofdimethylformamide. The mixture was heated with stirring at 125° C. for 6hours. Ethyl acetate (5 mL) was added to the cooled mixture, and thesolids were filtered and washed with ethyl acetate. The filtrate wasconcentrated under reduced pressure. The residual gum was trituratedwith 10 mL of water, and the resulting solid was filtered, washed wellwith water, and dried. This crude material was chromatographed byplacing a chloroform solution on a silica gel column wet withchloroform. The column was eluted with 1:1 (v/v) hexane:ethyl acetate,collecting the fractions that contain the Rf 0.25 spot on tlc (1:1hexane:ethyl acetate). Evaporation of the solvents gave a solid. Thesolid product was dissolved in about 0.5 mL of methylene chloride.Crystals develop. Petroleum ether (ca. 2 mL) was added, and the crystalswere filtered to provide 0.168 g (40%) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-ylideneamine, mp 198-200° C. Mass spectrum (CI) 351(M+1).

Analysis calculated for C₁₅ H₁₂ Cl₂ N₄ S: C, 51.29; H, 3.44; N, 15.95.Found: C, 51.31; H, 3.41; N, 15.73.

EXAMPLE 18 6-(2,6-Dichlorophenyl)-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl!-(3-diethylaminoprorpyl)-amine

A solution of 0.275 g (0.78 mmol) of the methylsulfanyl derivative fromExample 19 in 3 mL of N,N-diethylaminopropylamine was heated withstirring in a 135° C. oil bath (pot T=ca. 125° C.) for 16 hours. Theexcess amine was evaporated at reduced pressure, and the remaining oilwas dissolved in 10 mL of diethyl ether. The turbid solution wasclarified with "celite", filtered, and concentrated. The residue wastriturated with petroleum ether and filtered; wt 0.288 g (85% yield).Recrystallization from ethyl acetate-petroleum ether gave pure productidentified as 6-(2,6-dichlorophenyl)-7-imino-8-methyl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl!-(3-diethylaminopropyl)-amine; mp 154-156° C.

Mass spectrum (CI) 433 (M⁺). Analysis calculated for C₂₁ H₂₆ C₁₂ N₆.0.25H₂ O: C, 57.60; H, 6.10; N, 19.19. Found: C, 57.46; H, 5.85; N, 19.16.

EXAMPLE 19 6-(2,6-Dichlorophenyl)-8-methyl-7-oxo-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl!-(3-diethylaminopropyl)-amine

A solution of 0.111 g (0.25 mmol) of the imino derivative from Example20 in 5 mL of concentrated hydrochloric acid was heated at reflux for 6days. The aqueous acid was evaporated at reduced pressure, and theresidue was dissolved in 1.0 mL of water. Aqueous 10% potassiumcarbonate solution was added to completely precipitate a gum. Thesolvent was decanted, and the gum was dissolved in 15 mL of methylenechloride. The solution was dried over anhydrous potassium carbonate,filtered, and the filtrate was evaporated. The remaining gum wasdissolved in 0.5 mL of diethyl ether. The crystalline product whichdeveloped was filtered and dried to provide6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido2,3-d!pyrimidin-2-yl!-(3-diethylaminopropyl)-amine. Mass spectrum (CI)434 (M⁺).

EXAMPLE 20 2-Amino-6-phenyl-8-methyl-pyrido 2,3-d!pyrimidin-7(8H)-one

This compound was prepared from6-phenyl-7-imino-8-methyl-7,8-dihydro-pyrido 2,3-d!pyrimidin-2-ylamineof Example 9 by an acid hydrolysis procedure similar to that of Example14; mp 250-255° C.

EXAMPLE 21 2-Amino-6-(2,6-dichloroihenyl)-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-thion

A mixture of 0.321 g (1.0 mmol) of2-amino-6-(2,6-dichlorophenyl)-8-methyl-pyrido2,3-d!pyrimidin-7-(8H)-one from Example 12 and 0.404 g (1.0 mmol) ofLawesson's Reagent in 10 mL of pyridine was heated at reflux withstirring for 24 hours. The solvent was evaporated under reducedpressure, and the residue was triturated with 20 mL of water, filtered,and the cake washed well with water. Purification was by silica gelchromatography to afford the desired compound identified as2-amino-6-(2,6-dichlorophenyl)-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-thione.

EXAMPLE 22 N- 6-(2,6-Dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-ylidene!-acetamide ##STR37##

A mixture of 0.161 g (0.46 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-ylideneamine from Example 19 and 1.0 mL of aceticanhydride was heated to solution at the boiling point. After 2 minutesof reflux, the solution was concentrated to one-half volume, whereuponcrystals formed. The mixture was cooled, 2 mL of ether was added, andthe product was filtered and washed with ether; mp 229-231° C.

Mass spectrum (CI) 393 (M⁺). Analysis calculated for C₁₇ H₁₄ C₁₂ N₄ OS:C, 51.92; H, 3.59; N, 14.25. Found: C, 52.12; H, 3.62; N, 14.20.

EXAMPLE 23 N-6-(2,6-Dichlorophenyl)-2-(4-diethylaminobutylamino)-8-methyl-8H-pyrido2,3-d!-pyrimidin-7-ylidene!-acetamnide ##STR38##

A mixture of 0.112 g (0.29 mmol) of N-6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-ylidene!-acetamide of Example 28 and 1.0 mL (largeexcess) of 4-(diethyl-amino)butylamine was heated in a 135° C. oil bathwith stirring. After 1 hour, the solution was concentrated at reducedpressure, and the residue was triturated with 1 mL of ethyl acetate.Petroleum ether (1 mL) was added, and the product was filtered.

Mass spectrum (CI) 489 (M⁺).

EXAMPLE 24 2-Amino-6-(2,6-dichlorophenyl)-8-ethyl-pyrido2,3-d!-pyrimidin-7(8H)-one

To a suspension of NaH (60% in mineral oil, 27 mg) in 5 mL ofdimethylformamide was added 2-amino-6-(2,6-dichlorophenyl)-pyrido2,3-d!pyrimidin-7(8H)-one (172 mg, 0.56 mmol) from Example 11. Themixture was heated at 50° C. for 1 hour resulting in a clear solution.Ethyl iodide (60 μL, 0.75 mmol) was added, and the solution was stirredat 50° C. for 3.5 hours, cooled to room temperature, and poured into 30mL of ice water. The resulting precipitate was removed by filtration andpartitioned between ethyl acetate and water. The organic layer wasseparated and dried over MgSO4, filtered, and concentrated in vacuo.Flash chromatography, eluting with ethyl acetate, provided 104 mg (55%)of 2-amino-6-(2,6-dichlorophenyl)-8-ethyl-pyrido2,3-d!pyrimidin-7(8H)-one; mp 207-209° C.

Analysis calculated for C₁₅ H₁₂ C₁₂ N₄ O: C, 53.75; H, 3.61; N, 16.71.Found: C, 53.84; H, 3.67; N, 16.57.

EXAMPLE 25 2-Amino-6-(2,6-dichlorophenyl)-8-propyl-8H-pyrido2,3-d!-pyrimidin-7-one

To a suspension of NaH (60% in mineral oil, 31 mg) in 6 mL ofdimethylformamide was added 2-amino-6-(2,6-dichlorophenyl)-pyrido2,3-d!pyrimidin-7(8H)-one from Example 11 (205 mg, 0.67 mmol). Themixture was heated at 50° C. for 1 hour resulting in a clear solution.1-Iodopropane (100 μL, 1.03 mmol) was added, and the solution wasstirred at 50° C. for 10 minutes, then cooled to room temperature andpoured onto 40 mL of ice water. The resulting precipitate was removed byfiltration and washed with water. The residue was dried and purified byflash chromatography, eluting with 1:1 hexane:ethyl acetate to provide159 mg (68%) of 2-amino-6-(2,6-dichlorophenyl)-8-propyl-8H-pyrido2,3-d!pyrimidin-7-one; mp 196-197° C.

Analysis calculated for C₁₆ H₁₄ C₁₂ N₄ O: C, 55.03; H, 4.04; N. 16.04.Found: C, 55.28; H, 4.22; N, 15.81.

EXAMPLE 26 2-Amino-8-butyl-6-(2,6-dichlorophenyl)-8H-pyrido2,3-d!pyrimidin-7-one

To a suspension of NaH (60% in mineral oil, 34 mg) in 6 mL ofdimethylformamide was added 2-amino-6-(2,6-dichlorophenyl)-pyrido2,3-d!pyrimidin-7(8H)-one (202 mg, 0.66 mmol). The mixture was heated to50° C., resulting in a clear solution. 1-Iodobutane (105 μL, 0.92 mmol)was added, and the solution was stirred at 50° C. for 30 minutes, thencooled to room temperature and poured onto 40 mL of ice water. Theresulting precipitate was removed by filtration and washed with water.The residue was dried and purified by flash chromatography, eluting witha gradient of 1:1 hexane:ethyl acetate to all ethyl acetate to provide152 mg (64%) of 2-amino-8-butyl-6-(2,6-dichlorophenyl)-8H-pyrido2,3-d!pyrimidin-7-one; mp 202-205° C.

Analysis calculated for C₁₇ H₁₆ C₁₂ N₄ O.0.08 EtOAc: C, 56.18; H, 4.52;N, 15.13. Found: C, 56.39; H, 4.64; N, 14.99.

EXAMPLE 27 2-Amino-6-(2,6-dichlorophenyl)-8-isobutyl-8H-pyrido 23-d!pyrimidin-7-one

To a suspension of NaH (60% in mineral oil, 36 mg) in 8 mL ofdimethylformamide was added 2-amino-6-(2,6-dichlorophenyl)-pyrido2,3-d!pyrimidin-7(8H)-one (205 mg, 0.67 mmol). The mixture was heated at60° C. for 20 minutes resulting in a clear solution.1-Iodo-2-methylpropane (110 μL, 0.94 mmol) was added, and the solutionwas stirred at 50° C. for 30 minutes. An additional amount of1-iodo-2-methylpropane (40 μL, 0.34 mmol) was added, and the solutionwas stirred at 50° C. for 40 minutes, then cooled to room temperatureand poured onto 40 mL of ice water. The resulting precipitate wasremoved by filtration and washed with water. The gummy residue wasdissolved in ethyl acetate and washed with water. The organic layer wasdried over magnesium sulfate, filtered, and concentrated in vacuo. Theresulting solid was purified by flash chromatography, eluting with 1:1hexane:ethyl acetate to provide 123 mg (51%) of2-amino-6-(2,6-dichlorophenyl)-8-isobutyl-8H-pyrido2,3-d!pyrimidin-7-one; mp 193-195° C.

Analysis calculated for C₁₇ H₁₆ C₁₂ N₄ O: C, 56.21; H, 4.44; N, 15.42.Found: C, 56.60; H, 4.59; N, 15.11.

EXAMPLE 282-Amino-6-(2,6-dichlorophenyl)-8-(3-dimethylamino-propyl)-8H-pyrido2,3-d!pyrimidin-7-one

To a suspension of NaH (60% in mineral oil, 50 mg) in 8 mL ofdimethylformamide was added 2-amino-6-(2,6-dichlorophenyl)-pyrido2,3-d!pyrimidin-7(8H)-one (319 mg, 1.04 mmol). The mixture was heated at70° C. for 1.5 hours resulting in a clear solution. In a second flaskcontaining NaH (60% in mineral oil, 68 mg) in 6 mL of dimethylformamidewas added 3-dimethylaminopropyl chloride hydrochloride (248 mg, 1.56mmol). This suspension was stirred at room temperature for 30 minutes,then heated at 70° C. for 10 minutes and added to the above solution ofthe sodium salt of 2-amino-6-(2,6-dichlorophenyl)-pyrido2,3-d!pyrimidin-7(8H)-one. The resultant suspension was heated at 70° C.for 3 hours, then cooled to room temperature and filtered washing withethyl acetate. The filtrate was concentrated in vacuo and ethyl acetateand hexane were added. The resulting solid was collected by filtrationand dried in vacuo to provide 216 mg (53%) of2-amino-6-(2,6-dichlorophenyl)-8-(3-dimethyl-aminopropyl)-8H-pyrido2,3-d!pyrimidin-7-one; mp 136-141° C.

Analysis calculated for C₁₈ H₁₉ C₁₂ N₅ O: C, 55.11; H, 4.88; N, 17.85.Found: C, 55.07; H, 5.00; N, 17.53.

EXAMPLE 29 6-(2,6-Dichlorophenyl)-8-methyl-2-3-(4-methylpiperazin-1-yl)-propylamino!-8H-pyrido 2,3-d!pyrimidin-7-one

A mixture of 0.152 g (0.43 nmmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one of Example 37 and 1.0 g (6.40 mmol) of1-(3-aminopropyl)-4-methylpiperazine was heated with stirring in a 170°C. oil bath (pot T=ca. 160° C.) for 3 hours. The excess amine wasevaporated at reduced pressure. Water (5 mL) was added, and theseparated gum was extracted into 50 mL of 10% methylene chloride-ether.The organic phase was washed three times with 10 mL of water, dried(potassium carbonate), charcoaled, filtered, and concentrated. Theremaining gum was dissolved in 3 mL of ether. The crystals thatseparated on inducement were filtered and washed with ether; wt 0.033 g;mp 170-172° C.

Mass spectrum (CI) 461 (M⁺). Analysis calculated for C₂₂ H₂₆ C₁₂ N₆ O:C, 57.27; H, 5.68; N, 18.21. Found: C, 57.39; H, 5.70; N, 18.10.

EXAMPLE 30 6-(2,6-Dichlorophenyl)-2-methanesulfonyl-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one

A quantity of 0.346 g (1.00 mmol) of 50% to 60% m-chloroperbenzoic acid(assuming 50% peracid was present) was added at 25° C. to a stirredsolution of 0.165 g (0.47 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one of Example 37 in 15 mL of chloroform, and thesolution was stirred overnight. A quantity of 0.25 g (3.20 mmol) ofdimethylsulfoxide was added to reduce any excess peracid. After 15minutes, the chloroform solution was washed with 30 mL of saturatedsodium bicarbonate and then with water. The separated organic layer wasdried over sodium sulfate, filtered, and concentrated to ca. 5 mLvolume. Crystals separated. Added ca. 5 mL of petroleum ether andfiltered; wt 0.165 g (92%); mp>290° C.

Mass spectrum (CI) 384 (M⁺). Analysis calculated for C₁₅ H₁₁ C₁₂ N₃ O₃S: C, 46.89; H, 2.89; N, 10.94. Found: C, 47.14; H, 2.96; N, 10.87.

EXAMPLE 31 2-Benzylamino-6-(2,6-dichlorophenyl)-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one

A mixture of 0.165 g (0.47 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido-2,3-d!pyrimidin-7-one, 0.50 g (4.70 mmol) of benzylamine and 0.5 mL ofdimethylformamide was heated with stirring in a 120° C. oil bath. After5 minutes, solution was complete. After 5 hours, the excess amine anddimethylformamide were evaporated at reduced pressure, and the residuewas triturated with a solution of 1 mL of acetone and 2 mL of petroleumether. The tacky solid was filtered and recrystallized from ethylacetate/petroleum ether to give 0.092 g of pure product; mp 217-219° C.

Mass spectrum (CI) 411 (M⁺). Analysis calculated for C₁₇ H₁₄ C₁₂ N₄ O₅ :C, 61.33; H, 3.92; N, 13.62. Found: C, 61.30; H, 4.02; N, 13.59.

EXAMPLE 326-(2,6-Dichlororhenyl)-8-methyl-2-(3-moroholin-4-yl-propylamino)-8H-pyrido2,3-d!pyrimidin-7-one

A mixture of 0.165 g (0.47 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one, 1.00 g (6.90 mmol) of N-(3-aminopropyl)morpholineand 0.5 mL of dimethylformamide was heated with stirring in a 125° C.oil bath. After 2 minutes, solution was complete. After 1.5 hours, theexcess amine and dimethylformamide were evaporated at reduced pressureand the residue was triturated with 5 mL of water. The gum was dissolvedin 25 mL of ethyl acetate, and the solution was washed with 2×5 mL ofwater, dried (potassium carbonate), and concentrated. Upon dissolutionin 5 mL of ether, crystals of pure product separated; wt 0.101 g; mp140-142° C.

Mass spectrum (CI) 448 (M⁺). Analysis calculated for C₂₁ H₂₃ C₁₂ N₄ O₂ :C, 56.26; H, 5.17; N, 15.62. Found: C, 56.48; H, 5.24; N, 15.53.

EXAMPLE 33 6-(2,6-Dichlorophenyl)-8-methyl-2-(pyridin-2-ylmethyl)-amino!-8H-pyrido 2,3-d!pyrimidin-7-one

A mixture of 0.165 g (0.47 nmmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one, 1.08 g (10.0 mmol) of 2-(aminomethyl)pyridine and0.5 mL of dimethylformamide was heated with stirring in a 130° C. oilbath. After 2 minutes, solution was complete. After 2 hours, the excessamine and dimethylformamide were evaporated at reduced pressure. Ether(5 mL) was added to the residue. Crystals immediately developed. Water(5 mL) was added, and the entire mixture was filtered. The cake waswashed 5 mL of ether, 5 mL of water, and then dried; wt 0.164 g.Recrystallization from ethyl acetate gave 0.075 g of pure product; mp198-201° C.

Mass spectrum (CI) 412 (M⁺). Analysis calculated for C₂₀ H₁₅ C₁₂ N₅ O:C, 58.27; H, 3.67; N, 16.99. Found: C, 58.36; H, 3.82; N, 16.82.

EXAMPLE 34 6-(2,6-Dichlorophenyl)-8-methyl-2-(pyridin-3-ylmethyl)-amino!-8H-pyrido 2,3-d!pyrimidin-7-one

This compound was prepared by a procedure similar to that described inExample 33 starting with 0.165 g (0.47 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one of Example 37 and 1.08 g (10.0 mmol) of3-(aminomethyl)-pyridine yielding 0.053 g of pure product; mp 224-226°C.

Mass spectrum (CI) 412 (M⁺) Analysis calculated for C₂₀ H₁₅ C₁₂ N₅ O: C,58.27; H, 3.67; N, 16.99. Found: C, 58.36; H, 3.78; N, 16.79.

EXAMPLE 356-(2,6-Dichlorophenyl)-8-methyl-2-(2-pyridin-2-yl-ethylamino)-8H-pyrido2,3-d!pyrimidin-7-one

This compound was prepared by a procedure similar to that described inExample 33 starting with 0.165 g (0.47 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one of Example 37 and 1.00 g (8.20 mmol) of2-(2-aminoethyl)-pyridine yielding 0.082 g of pure product; mp 173-174°C.

Mass spectrum (CI) 426 (M⁺). Analysis calculated for C₂₁ H₁₇ Cl₂ N₅ O:C, 59.17; H, 4.02; N, 16.43. Found: C, 59.28; H, 4.11; N, 16.29.

EXAMPLE 36 6-(2,6-Dichlorophenyl)-2-{3-4-(2-methoxyohenyl)-piperazin-1-yl!-propylamino}-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one

This compound was prepared by a procedure similar to that described inExample 33 starting with 0.165 g (0.47 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one of Example 37 and 1.00 g (4.00 mmol) of1-(3-aminopropyl)-4-(2-methoxyphenyl)piperazine yielding 0.103 g of pureproduct; mp 187-188° C.

Mass spectrum (CI) 553 (M⁺). Analysis calculated for C₂₈ H₃₀ Cl₂ N₆ O₂ :C, 60.76; H, 5.46; N, 15.18. Found: C, 61.04; H, 5.41; N, 15.20.

EXAMPLE 376-(2,6-Dichlorophenyl)-8-methyl-2-(pyridin-4-ylamino)-8H-pyrido2,3-d!pyrimidin-7-one

A mixture of 0.550 g (1.25 mmol) of6-(2,6-dichlorophenyl)-2-methanesulfonyl-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one and 1.50 g (16.0 mmol) of 4-aminopyridine washeated, with stirring, in a 150° C. oil bath. The resulting solution washeated for 10 minutes and cooled to room temperature. The hardened meltwas triturated with 3 mL of methanol. After 24 hours of standing, thegranular solid that developed was filtered and washed with 2 mL ofmethanol and 2 mL of ether; wt 0.471 g. The hydrochloride salt wasprepared as follows: The above crude base was suspended in 5 mL ofmethanol. With stirring, 1 mL of 2N hydrochloric acid was added to givea complete solution. Additional hydrochloric acid was added until thesolution was slightly turbid. The crystals that separated on inducementwere filtered and washed with 10 mL of 10% methanol-ether and thenether; wt 0.485 g. Recrystallization from methanol/ether gave purecrystalline product; wt 0.405 g; mp 338-340° C.

Mass spectrum (CI) 398 (M⁺). Analysis calculated for C₁₉ H₁₃ Cl₂ N₅O.HCl.H₂ O: C, 50.40; H, 3.56; N, 15.47. Found: C, 50.78; H, 3.18; N,15.50.

EXAMPLE 38 6-(2,6-Dichlorophenyl)-8-methyl-2-4-(4-methylpiperazin-1-yl)-butylamino!-8H-pyrido 2,3-d!-pyrimidin-7-one

A mixture of 0.152 g (0.43 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one and 0.50 g (2.90 mmol) of1-(4-aminobutyl)-4-methylpiperazine was heated with stirring in a 170°C. oil bath. After 2 minutes, solution was complete. After 2 hours, thesolution was cooled to room temperature, and the dark gum was dissolvedin 25 mL of ether. The solution was washed with 4×5 mL of water. Most ofthe color went into the water wash. The ether solution was dried overpotassium carbonate, filtered, and concentrated to ca. 2 mL volume. Thecrystals that separated on inducement were filtered and washed withether; wt 0.063 g; mp 130-132° C.;

Mass spectrum (CI) 475 (M⁺). Analysis calculated for C₂₃ H₂₈ Cl₂ N₆ O:C, 58.11; H, 5.94; N, 17.68. Found: C, 58.39; H, 5.99; N, 17.53.

EXAMPLE 39 6-(2,6-Dichlorophenyl)-8-ethyl-2-3-(4-methyl-piperazin-1-yl)-propylamino!-8H-pyrido 2,3-d!pyrimidin-7-one

A mixture of 0.150 g (0.41 mmol) of6-(2,6-dichlorophenyl)-8-ethyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one and 0.500 g (3.20 mmol) of1-(3-aminopropyl)-4-methylpiperazine was heated in a 180° C. oil bath.The resulting solution was heated for 0.5 hour. Most of the excess aminewas evaporated at reduced pressure. The remaining gum was trituratedwith 2 mL of water and decanted. The gum was dissolved in 15 mL ofether. The solution was washed with 3×5 mL of water, dried (sodiumsulfate), filtered, and concentrated to 2 mL volume. Petroleum ether wasadded until slight turbidity. The crystals that separated on inducementwere filtered and washed with 2 mL of 75% ether/petroleum ether; wt 0.90g; mp 126-128° C.

Mass spectrum (CI) 475 (M⁺) Analysis calculated for C₂₃ H₂₈ Cl₂ N₆ O.0.4H₂ O: C, 57.24; H, 6.01; N, 17.42. Found: C, 57.33; H, 6.04; N, 17.07.

EXAMPLE 406-(2,6-Dichlorophenyl)-2-(2-methoxyphenylamino)-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one

A mixture of 0.113 g (0.29 mmol) of6-(2,6-dichlorophenyl)-2-methanesulfonyl-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one and 0.50 g (4.10 mmol) of 2-methoxyaniline washeated, with stirring, in a 175° C. oil bath. The resulting solution washeated for 5 minutes and cooled to room temperature. Ether (2 mL) wasadded. The crystals that developed were filtered and washed with 1 mL ofether; wt 0.070 g. The solid was purified to remove dark colors bysilica gel chromatography, eluting with chloroform. Recrystallizationfrom ether gave pure product; wt 0.029 g; mp 200-201° C.

Mass spectrum (CI) 427 (M⁺). Analysis calculated for C₂₁ H₁₆ Cl₂ N₄ O₂ :C, 59.03; H, 3.77; N, 13.11. Found: C, 59.09; H, 3.87; N, 13.02.

EXAMPLE 416-(2,6-Dichlorophenyl)-8-methyl-2-(pyridin-3-ylamino)-8H-pyrido2,3-d!pyrimidin-7-one

A mixture of 0.165 g (0.47 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one, 0.500 g (5.30 mmol) of 3-aminopyridine base, and0.066 g (0.50 mmol) of 3-aminopyridine hydrochloride was heated withstirring in a 210° C. oil bath for 1 hour. Water (5 mL) was added to thecooled reaction mixture to precipitate a solid. The solid was filtered,washed well with water, and dried; wt 0.147 g. Purification was effectedby silica gel chromatography by eluting with chloroform and then ethylacetate. The ethyl acetate eluent containing the pure product wasconcentrated to 2 mL volume. The crystals that separated on inducementwere filtered and washed with 0.5 mL of ethyl acetate and 1 mL of ether;wt 0.810 g; mp 247-248° C.

Mass spectrum (CI) 398 (M⁺). Analysis calculated for C₁₉ H₁₃ Cl₂ N₅ O:C, 57.30; H, 3.29; N, 17.59. Found: C, 57.33; H, 3.38; N, 17.43.

EXAMPLE 42 6-(2,6-Dichlorophenyl)-2-4-(2-diethylaminoethoxy)-phenylamino!-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one

A mixture of 0.155 g (0.40 mmol) of6-(2,6-dichlorophenyl)-8-methyl-2-methylsulfanyl-8H-pyrido2,3-d!pyrimidin-7-one, 0.167 g (0.80 mmol) of4-(2-diethylaminoethoxy)aniline and 1 mL of (2-methoxyethyl)ether (bp162° C.) was heated with stirring in a 150° C. oil bath. All solidgradually dissolved over a period of 10 minutes. The solution was heatedanother 10 minutes and cooled to 100° C. Water was added dropwise untilslight turbidity. The crystals that separated on inducement werefiltered, washed with 0.5 mL of ether and 2 mL of water, and dried; wt0.105 g. Purification was effected by chromatography eluting withchloroform, then ethyl acetate and finally 10% methanol/chloroform toobtain fraction with pure product. The eluent was evaporated to dryness.The remaining amorphous solid was dissolved in 1 mL of warm ethylacetate. The crystals that separated on inducement were filtered andwashed sparingly with ethyl acetate and ether; wt 0.042 g; mp 141-143°C.

Mass spectrum (CI) 512 (M⁺). Analysis calculated for C₂₆ H₂₇ Cl₂ N₅ O₂ :C, 60.94; H, 5.31; N, 13.67. Found: C, 60.96; H, 5.36; N, 13.52.

EXAMPLE 43 6-(2,6-Dichlorophenyl)-2- 4-2-(diethylamino)ethoxyl-thenyl!aminol-8-methyl-pyridor2,3-d!pyrimidin-7(8H)-one, N.sup.ω -oxide ##STR39##

To a stirred solution of 6-(2,6-dichlorophenyl)-2-4-(2-diethylamino-ethoxy)-phenylamino!-8-methyl-8H-pyrido2,3-d!pyrimidin-7-one (0.5 g, 0.98 mmol) in chloroform (15 mL) was addeda solution of 2-(phenylsulfonyl)-3-phenyloxaziridine (0.25 g, 0.98 mmol)dropwise at room temperature. The reaction mixture was stirred for 3hours at 240° C. The solvent was removed by evaporation under reducedpressure. The residue was dissolved in a 30 mL of ethylacetate/methanol/triethylamine (86:10:4), and the solution was appliedto a radial chromatography apparatus. The compound was purified byradial chromatography eluting with a gradient of ethylacetate/methanol/triethylamine (86:10:2) to ethylacetate/methanol/triethylene (86:10:4). After combining fractionscontaining the major component and removing the solvent by evaporation,the product was recrystallized from 15 mL of isopropyl alcohol to give0.25 g of the titled compound: mp 180-181° C.

Analysis calculated for C₂₆ H₂₇ N₅ O₃ Cl₂.0.08 H₂ O: C, 58.92; H, 5.44;N, 13.21. Found: C, 58.53; H, 5.12; N, 12.98.

EXAMPLE 44-50

By following the general procedure of Example 43, the following N-oxidescan be prepared by oxidizing the corresponding tertiary amine:

6-(3,5-dimethoxyphenyl)-2- 4-2-diethylamino)ethoxy!-phenylamino!-8-ethyl-pyrido2,3-d!pyrimidin-7(8H)-one, N.sup.ω -oxide;

6-(3,5-dimethylthiophenyl)-2-4-(methylethylamino)phenyl!methyl!amino!-8-cyclopropyl-pyrido2,3-d!pyrimidin-7(8H)-thione, N.sup.ω -oxide;

6-(3-methoxy-5-ethoxyphenyl)-2-2-(methylethylamino)ethyl!amino!-8-n-butyl-pyrido2,3-d!pyrimidin-7(8H)-one, N.sup.ω -oxide;

6-(4-ethyl-5-methoxyphenyl)-2- 4-4-methyl-1-piperazinyl!butyl!amino!-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-one, N.sup.ω -oxide, having the formula;##STR40## and a compound of the formula ##STR41##

EXAMPLE 51 (8-Ethyl-7-imino-6-naphthalen-2-yl-7,8-dihydro-pyrido 23-d!pyrimidin-2-yl)-phenylamine

To a suspension of NaH (60% in mineral oil, 27 mg) in 5 mL of2-ethoxyethanol was added 2-naphthyl-acetonitrile (227 mg, 1.36 mmol).After stirring for 5 minutes at room temperature,4-ethylamino-2-phenylamino-pyrimidine-5-carbaldehyde (300 mg, 1.24 mmol)was added and the reaction heated at 110° C. for 1 hour, resulting in adark brown solution. Upon cooling, the solution was poured into 30 mL ofwater which caused precipitation. The resulting precipitate was removedby filtration and washed with water. The crude product was purified byflash chromatography, eluting with 5% methanol/methylene chloride,followed by 10% methanol/methylene chloride. Concentration of productfractions yielded 400 mg (82%) of yellow solid,(8-ethyl-7-imino-6-naphthalen-2-yl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl)-phenylamine; mp 236-242° C.

Mass spectrum (CI) 392 (M⁺). Analysis calculated for C₂₅ H₂₁ N₅ : C,76.70; H 5.41; N, 17.89. Found: C, 75.58; H 5.49; N, 17.58.

EXAMPLE 52 8-Ethyl-6-naphthalen-2-yl-2-phenylamino-8H-pyrido2,3-d!pyrimidin-7-one

(8-Ethyl-7-imino-6-naphthalen-2-yl-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl)-phenylamine (150 mg) was added to 1 mL of aceticanhydride and heated at reflux for 2 minutes. The reaction was cooledand concentrated, resulting in an oil which was heated at reflux with 10mL of 6N HCl for 10 minutes. The reaction was cooled, and 40 mL of waterwas added causing precipitation. The precipitate was removed byfiltration and washed with water. The resulting solid was dried in avacuum oven to provide 8-ethyl-6-naphthalen-2-yl-2-phenylamino-8H-pyrido2,3-d!-pyrimidin-7-one; mp 254-256° C.

Mass spectrum (CI) 393 (M⁺). Analysis calculated for C₂₅ H₂₀ N₄ O.HCl:C, 70.00; H, 4.94; N, 13.06. Found: C, 68.61; H, 4.97; N, 12.83.

EXAMPLE 53 (6-Biphenyl-4-yl-8-ethyl-7-imino-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl)-phenylamine

To a suspension of NaH (60% in mineral oil, 27 mg) in 5 mL of2-ethoxyethanol was added 4-biphenylaceto-nitrile (263 mg, 1.36 mmol).After stirring for 5 minutes at room temperature,4-ethylamino-2-phenylamino-pyrimidine-5-carboxaldehyde (300 mg, 1.24mmol) was added and the reaction heated at 110° C. for 1 hour, resultingin a dark brown solution. Upon cooling, the solution was poured intowater which caused precipitation. The resulting precipitate was removedby filtration and washed with water. The crude product was purified byflash chromatography, eluting with 5% methanol/methylene chloride,followed by 10% methanol/methylene chloride. Concentration of productfractions yielded 427 mg (83%) of(6-biphenyl-4-yl-8-ethyl-7-imino-7,8-dihydro-pyrido2,3-d!pyrimidin-2-yl)-phenylamine; mp 245-249° C.

Mass spectrum (CI) 418 (M⁺). Analysis calculated for C₂₇ H₂₃ N₅ : C,77.67; H, 5.55; N, 16.78. Found: C, 76.16; H, 5.54; N, 16.36.

The compounds of Formula I are valuable inhibitors of protein tyrosinekinases and possess therapeutic value as cellular antiproliferativeagents for the treatment of proliferative disorders. These compounds arepotent inhibitors of one or more of the protein kinases, PDGF, FGF, EGF,viral-src (V-src), and cellular-src (C-src). The invention compounds arethus useful in treating atherosclerosis, restenosis, and cancer.Specific tumors to be treated with the compounds include small-cell lungcarcinoma such as that described in An. Rev. Respir. Dis., 142:554-556(1990); human breast cancer as described in Cancer Research,52:4773-4778 (1992); low grade human bladder carcinomas of the typedescribed in Cancer Research, 52:1457-1462 (1992); human colorectalcancer as discussed in J. Clin. Invest., 91:53-60 (1993); and in J.Sura. Res., 54:293-294 (1993).

The compounds of this invention have been evaluated in standard assayswhich are utilized to determine inhibition of tyrosine kinases. Theassays were conducted as follows:

Purification of Epidermal Growth Factor Receptor Tyrosine Kinase

Human EGF receptor tyrosine kinase was isolated from A431 epidermoidcarcinoma cells by the following methods. Cells were grown in rollerbottles in 50% Dulbecco's Modified Eagle medium and 50% HAM F-12nutrient media (Gibco) containing 10% fetal calf serum. Approximately10⁹ cells were lysed in two volumes of buffer containing 20 mM 2-(4N-2-hydroxymethyl!-piperazin-1-yl)ethanesulfonic acid, pH 7.4, 5 mMethylene glycol bis(2-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1%Triton X-100, 10% glycerol, 0.1 mM sodium orthovanadate, 5 mM sodiumfluoride, 4 mM pyrophosphate, 4 mM benzamide, 1 mM dithiothreitol, 80μg/mL aprotinin, 40 μg/mL leupeptin, and 1 mM phenylmethylsulfonylfluoride. After centrifugation at 25,000×g for 10 minutes, thesupernatant was equilibrated for 2 hours at 4° C. with 10 mL of wheatgerm agglutinin sepharose that was previously equilibrated with 50 mM4Hepes, 10% glycerol, 0.1% Triton X-100 and 150 mrM NaCl, pH 7.5,(equilibration buffer). Contaminating proteins were washed from theresin with 1 M NaCl in equilibration buffer, and the enzyme was elutedwith 0.5M N-acetyl-1-D-glucosamine in equilibration buffer.

Determination of IC₅₀ Values

Enzyme assays for IC₅₀ determinations were performed in a total volumeof 0.1 mL, containing 25 mM Hepes, pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂, 50 μMsodium vanadate, 5-10 ng of EGF receptor tyrosine kinase, 200 μM of asubstrate peptide (Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr⁴⁷²-Glu-Glu-Val-NH₂, derived from the amino acid (Tyr⁴⁷² has been shown tobe 1 of 4 tyrosines in PLC-g that are phosphorylated by the EGF receptortyrosine kinase (Wahl M. I., et al., J. Biol. Chem., 265:3944-3948(1990)), and peptides derived from the enzyme sequence surrounding thissite are excellent substrates for the enzyme), 10 μM ATP containing 1μCi of ³² P!ATP and incubated for 10 minutes at room temperature. Thereaction was terminated by the addition of 2 mL of 75 mM phosphoric acidand passed through a 2.5-cm phosphocellulose filter disc to bind thepeptide. The filter was washed 5 times with 75 mM phosphoric acid andplaced in a vial along with 5 mL of scintillation fluid (Ready gelBeckman).

PDGF and FGF Receptor Tyrosine Kinase Assays

Full length cDNAs for the mouse PDGF-β and human FGF-1 (flg) receptortyrosine kinases were obtained from J. Escobedo and prepared asdescribed in J. Biol. Chem., 262:1482-1487 (1991), and PCR primers weredesigned to amplify a fragment of DNA that codes for the intracellulartyrosine kinase domain. The fragment was melded into a baculovirusvector, cotransfected with AcMNPV DNA, and the recombinant virusisolated. SF9 insect cells were infected with the virus to overexpressthe protein, and the cell lysate was used for the assay. The assay wasperformed in 96-well plates (100 μL/incubation/well), and conditionswere optimized to measure the incorporation of ³² p from γ³² P-ATP intoa glutamate-tyrosine co-polymer substrate. Briefly, to each well wasadded 82.5 μL of incubation buffer containing 25 mM Hepes (pH 7.0), 150mM NaCl, 0.1% Triton X-100, 0.2 mM PMSF, 0.2 mM Na₃ VO₄, 10 mM MnCl₂,and 750 μg/mL of Poly (4:1) glutamate-tyrosine followed by 2.5 μL ofinhibitor and 5 μL of enzyme lysate (7.5 μg/μL FGF-TK or 6.0 μg/μLPDGF-TK) to initiate the reaction. Following a 10 minute incubation at25° C., 10 μL of γ³² P-ATP (0.4 μCi plus 50 μM ATP) was added to eachwell and samples were incubated for an additional 10 minutes at 25° C.The reaction was terminated by the addition of 100 μL of 30%trichloroacetic acid (TCA) containing 20 mM sodium pyrophosphate andprecipitation of material onto glass fiber filter mats (Wallac). Filterswere washed 3 times with 15% TCA containing 100 mM sodium pyrophosphateand the radioactivity retained on the filters counted in a Wallac 1250Betaplate reader. Nonspecific activity was defined as radioactivityretained on the filters following incubation of samples with bufferalone (no enzyme). Specific enzymatic activity was defined as totalactivity (enzyme plus buffer) minus nonspecific activity. Theconcentration of a compound that inhibited specific activity by 50%(IC₅₀) was determined based on the inhibition curve.

V-src and C-src Kinase Assays

V-src or C-src kinase is purified from baculovirus infected insect celllysates using an antipeptide monoclonal antibody directed against theN-terminal 2-17 amino acids. The antibody, covalently linked to 0.65-μmlatex beads, is added to a suspension of insect cell lysis buffercomprised of 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM DTT, 1% NP-40, 2 mMEGTA, 1 mM sodium vanadate, 1 mM PMSF, 1 μg/mL each of leupeptin,pepstatin, and aprotinin. Insect cell lysate containing either the C-srcor V-src protein is incubated with these beads for 3-4 hours at 4° C.with rotation. At the end of the lysate incubation, the beads are rinsed3 times in lysis buffer, resuspended in lysis buffer containing 10%glycerol, and frozen. These latex beads are thawed, rinsed 3 times inassay buffer which is comprised of 40 mM tris pH 7.5, 5 mM MgCl₂, andsuspended in the same buffer. In a Millipore 96-well plate with a 0.65μm polyvinylidine membrane bottom are added the reaction components:10-μL V-src or C-src beads, 10 μL of 2.5 mg/mL poly GluTyr substrate, 5μm ATP containing 0.2 μCi labeled ³² P-ATP, 5 μL DMSO containinginhibitors or as a solvent control, and buffer to make the final volume125 μL. The reaction is started at room temperature by addition of theATP and quenched 10 minutes later by the addition of 125 μL of 30% TCA,0.1M sodium pyrophosphate for 5 minutes on ice. The plate is thenfiltered and the wells washed with two 250-μL aliquots of 15% TCA, 0.1Mpyrophosphate. The filters are punched, counted in a liquidscintillation counter, and the data examined for inhibitory activity incomparison to a known inhibitor such as erbstatin. The method isdescribed more fully in J. Med. Chem., 37:598-609 (1994).

Cell Culture

Rat aorta smooth muscle cells (RASMC) were isolated from the thoracicaorta of rats and explanted according to the method of Ross, J. Cell.Biol., 30:172-186 (1971). Cells were grown in Dulbecco's modifiedEagle's medium (DMEM, Gibco) containing 10% fetal calf serum (FBS,Hyclone, Logan, Utah), 1% glutamine (Gibco) and 1%penicillin/streptomycin (Gibco). Cells were identified as smooth musclecells by their "hill and valley" growth pattern and by fluorescentstaining with a monoclonal antibody specific for SMC ∝-actin (Sigma).RASMC were used between passages 5 and 20 for all experiments. Testcompounds were prepared in dimethylsulfoxide (DMSO) in order to achieveconsistency in the vehicle and to ensure compound solubility.Appropriate DMSO controls were simultaneously evaluated with the testcompounds.

³ H!-Thymidine Incorporation Assay

RASMC were plated into a 24-well plate (30,000 cells/well) in DMEM with10% FBS. After 4 days, cells reached confluence and were made quiescentby incubation in DMEM/F12 medium (Gibco) containing 0.2% FBS for another2 days. DNA synthesis was induced by incubating cells for 22 hours witheither PDGF-BB, bFGF, or FBS, plus test compound in 0.5 mL/wellserum-substituted medium (DMEM/F12+1% CPSR-2 from Sigma). After 18hours, 0.25 μCi/well ³ H!-thymidine was added. Four hours later, theincubation was stopped by removing the radioactive media, washing thecells twice with 1 mL cold phosphate-buffered saline, and then washing 2times with cold 5% trichloroacetic acid. The acid-insoluble fraction waslysed in 0.75 mL 0.25N NaOH and the radioactivity determined by liquidscintillation counting. IC₅₀ values were determined graphically.

PDGF Receptor Autophosphorylation

RASMC were grown to confluency in 100 mm dishes. Growth medium wasremoved and replaced with serum-free medium and cells were incubated at37° C. for an additional 24 hours. Test compounds were then addeddirectly to the medium and cells incubated for an additional 2 hours.After 2 hours, PDGF-BB was added at a final concentration of 30 ng/mLfor 5 minutes at 37° C. to stimulated autophosphorylation of the PDGFreceptor. Following growth factor treatment, the medium was removed, andcells were washed with cold phosphate-buffered saline and immediatelylysed with 1 mL of lysis buffer (50 mM HEPES pH 7.5!, 150 mM NaCl, 10%glycerol, 1% Triton X-100, 1 mM EDTA, 1 mMEGTA, 50 mM NaF, 1 mM sodiumorthovanadate, 30 mM p-nitrophenyl phosphate, 10 mM sodiumpyrophosphate, 1 mM phenylmethyl sulfonyl fluoride, 10 μg/mL aprotinin,and 10 μg/mL leupeptin). Lysates were centrifuged at 10,000×g for 10minutes. Supernatants were incubated with 10 μL of rabbit anti-humanPDGF type AB receptor antibody (1:1000) for 2 hours. Following theincubation, protein-A-sepharose beads were added for 2 hours withcontinuous mixing, and immune complexes bound to the beads washed fourtimes with 1 mL lysis wash buffer. Immune complexes were solubilized in30 μL of Laemmli sample buffer and electrophoresed in 4-20% SDSpolyacrylamide gels. Following electrophoresis, separated proteins weretransferred to nitrocellulose and immunoblotted withanti-phosphotyrosine antiserum. Following incubation with ¹²⁵!I-protein-A, the levels tyrosine phosphorylated proteins were detectedby phosphorimage analysis and protein bands quantitated viadensitometry. IC₅₀ values were generated from the densitometric data.

Transplanted Tumor Assay

Several of the invention compounds will increase the life span ofanimals infected with transplanted tumors. F1 hybrid mice were used inthe assay. The mice receive ascites fluid or dilutions of tumor brei onDay 0. A sample of the inocula is incubated in thioglycolate media as acheck for gross contamination of the tumor material. After all testanimals are inoculated with tumors, they are randomized for the assay.Control animals receive vehicle, while treated animals receive aninvention compound dissolved in the vehicle, generally by infusion bytail tether. All animals are monitored daily for acute toxicity andother clinical signs. Survival is checked daily for the control groupand treated group. The assay generally is continued for 60 days, atwhich time all surviving animals are euthanized.

The following Table 1 presents biological data for representativecompounds of the invention when analyzed in the foregoing assays.

                  TABLE 1                                                         ______________________________________                                        Inhibition of Protein Tyrosine Kinases                                        (IC.sub.50  μM or % Inhibition at 50 μM)                                Example   PDGFr-TK  FGFr-TK    C-src TK                                                                             EGF-FL                                  ______________________________________                                        42        4.49      0.8        0.588  82%                                     43        0.07      0.063      0.015  --                                      ______________________________________                                    

As noted above, the compounds of Formula I are useful for treatingcancer and other proliferative diseases such as psoriasis, restenosis,and atherosclerosis.

The invention compounds are especially useful for treating restenosisfollowing balloon angioplasty of occluded arteries. Restenosis occurs inabout 40% of individuals undergoing angioplasty of calcified arteriesand is a major problem associated with this form of treatment ofpatients suffering from such cardiac condition. The invention compoundsdemonstrate good activity when evaluated in standard tests such asdescribed below.

Cyclin-Dependent Kinase Assays (cdk2/cyclinE, cdk2/cyclinA,cdc2/cyclinB)

Enzyme assays for IC₅₀ determinations and kinetic evaluation wereperformed in a 96-well filter plate (Millipore MADVN6550) in a totalvolume of 0.1 mL of 20 mM TRIS (Tris hydroxymethyl!aminomethane), pH7.4, 50 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl₂, 12 μM ATP containing0.25 μCi of ³² P!ATP, 20 ng of enzyme (either cdk2/cyclinE,cdk2/cyclinA, or cdc2/cyclinB), 1 μg retinoblastoma and appropriatedilutions of the particular invention compound. All components exceptthe ATP were added to the wells, and the plate was placed on a platemixer for 2 minutes. The reaction was begun by addition of ³² P!ATP, andthe plate was incubated at 25° C. for 15 minutes. The reaction wasterminated by addition of 0.1 mL of 20% trichloroacetic acid (TCA). Theplate was kept at 4° C. for at least 1 hour to allow the substrate toprecipitate. The wells were then washed five times with 0.2 mL of 10%TCA and ³² P incorporation determined using a beta plate counter (WallacInc., Gaithersburg, Md.).

Balloon Angioplasty of Rat Carotid Arteries

Male Sprague-Dawley rats (350-450 g) are divided into 2 treatmentgroups: 1 group of rats (n=10) are treated with drug (100 mg/kg PO, BID)and the second group received vehicle (2 mL/kg PO, BID (n=10)). Allanimals were pretreated for 2 days prior to surgery and continued toreceive daily drug treatment postinjury until sacrificed.

Balloon injury in rat carotid arteries were performed according to thefollowing protocol. Rats were anesthetized with Telazol (0.1 mL/100 gIM), and the carotid artery exposed via an anterior mid-line incision onthe neck. The carotid artery was isolated at the bifurcation of theinternal and external carotid arteries. A 2F embolectomy catheter wasinserted in the external carotid artery and advanced down the commoncarotid to the level of the aortic arch. The balloon was inflated andthe catheter is dragged back to the point of entry and then deflated.This procedure is repeated 2 more times. The embolectomy catheter wasthen removed and the external carotid artery was ligated leaving flowintact through the internal carotid artery. Surgical incisions wereclosed, and the animal was allowed to recover from anesthesia beforebeing returned to its home cage.

At various time points postinjury animals were euthanized with CO₂inhalation, and the carotid artery was perfusion fixed and processed forhistologic examination. Morphologic determination of lesion size wasmade by measuring the area of the carotid artery intima expressed as aratio of the media in individual animals. Up to 16 sections wereprepared from each animal to give a uniform representation of lesionsize down the length of the carotid artery. The cross-sectional areas ofthe blood vessels were quantified using an image analysis program fromPrinceton Gamma Tech (Princeton, N.J.).

The compounds of the present invention can be formulated andadministered in a wide variety of oral and parenteral dosage forms,including transdermal and rectal administration. It will be recognizedto those skilled in the art that the following dosage forms may compriseas the active component, either a compound of Formula I or acorresponding pharmaceutically acceptable salt or solvate of a compoundof Formula I.

A further embodiment of this invention is a pharmaceutical formulationcomprising a compound of Formula I together with a pharmaceuticallyacceptable carrier, diluent, or excipient therefor. For preparingpharmaceutical compositions with the compounds of the present invention,pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier can beone or more substances which may also act as diluents, flavoring agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier is a finely divided solid such as talc or starchwhich is in a mixture with the finely divided active component.

In tablets, the active component is mixed with the carrier having thenecessary binding properties in suitable proportions and compacted inthe shape and size desired.

The formulations of this invention preferably contain from about 5% toabout 70% or more of the active compound. Suitable carriers includemagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like. Apreferred form for oral use are capsules, which include the formulationof the active compound with encapsulating material as a carrierproviding a capsule in which the active component with or without othercarriers, is surrounded by a carrier, which is thus in association withit. Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogenous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water-propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution, isotonic saline, 5% aqueous glucose, andthe like.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizing and thickening agents as desired.

Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with a viscous material, suchas natural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like. Waxes, polymers, microparticles, and the like can be utilizedto prepare sustained-release dosage forms. Also, osmotic pumps can beemployed to deliver the active compound uniformally over a prolongedperiod.

The pharmaceutical preparations of the invention are preferably in unitdosage form. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component. The unitdosage form can be a packaged preparation, the package containingdiscrete quantities of preparation, such as packeted tablets, capsules,and powders in vials or ampoules. Also, the unit dosage form can be acapsules, tablet, cachet, or lozenge itself, or it can be theappropriate number of any of these in packaged form.

The therapeutically effective dose of a compound of Formula I willgenerally be from about 1 mg to about 100 mg/kg of body weight per day.Typical adult doses will be about 50 to about 800 mg per day. Thequantity of active component in a unit dose preparation may be varied oradjusted from about 0.1 mg to about 500 mg, preferably about 0.5 mg to100 mg according to the particular application and the potency of theactive component. The composition can, if desired, also contain othercompatible therapeutic agents. A subject in need of treatment with acompound of Formula I will be administered a dosage of about 1 to about500 mg per day, either singly or in multiple doses over a 24-hourperiod.

EXAMPLE 54

A pharmaceutical formulation in the form of hard gelatin capsules fororal administration are prepared using the following ingredients:

    ______________________________________                                                              Quantity                                                                      (mg/capsule)                                            ______________________________________                                        Active compound       250                                                     Starch powder         200                                                     Magnesium stearate    10                                                      Total                 460    mg                                               ______________________________________                                    

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities. A typical active ingredient is the N-oxide of Example43. The composition is administered from 2 to 4 times a day fortreatment of postsurgical restenosis.

EXAMPLE

    ______________________________________                                        Formulation for Oral Suspension                                               Ingredient                Amount                                              ______________________________________                                        2-(2-dimethylamino-cyclopropylamino-N.sup.ω -                                                     500    mg                                           oxide)-6-(2-bromo-4-methoxy-5-ethyl-                                          thiophenyl)-8-n-hexyl-pyrido 2,3-d!-                                          pyrimidine-7(8H)-one                                                          Sorbitol solution (70% N.F.)                                                                            40     ml                                           Sodium benzoate           150    mg                                           Saccharin                 10     mg                                           Cherry Flavor             50     mg                                           Distilled water q.s. ad   100    mL                                           ______________________________________                                    

The sorbitol solution is added to 40 mL of distilled water and thepyridopyrimidine is suspended therein. The saccharin, sodium benzoate,and flavoring are added and dissolved. The volume is adjusted to 100 mLwith distilled water. Each milliliter of syrup contains 5 mg of activeingredient.

EXAMPLE

    ______________________________________                                        Active ingredient       60     mg                                             Starch                  45     mg                                             Microcrystalline cellulose                                                                            35     mg                                             Polyvinylpyrrolidone    4      mg                                             (as 10% solution in water)                                                    Sodium carboxymethyl starch                                                                           4.5    mg                                             Magnesium stearate      0.5    mg                                             Talc                    1.0    mg                                             Total                   150    mg                                             ______________________________________                                    

The active ingredients, starch, and cellulose, are passed through a No.45 mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders and then passedthrough a No. 14 mesh U.S. sieve. The granules are dried at 50° C. to60° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

A typical active ingredient utilized in the above preparation is thecompound of Example 43.

EXAMPLE 57

Slow Release Preparation

Five hundred milligrams of 6-(2,6-dichlorophenyl)-2-4-(2-diethylaminoethoxy)-phenylamino-N.sup..omega.-oxide!-8-methoxy-8H-pyrido 2,3-d!pyrimidin-7-one hydrochloride wasplaced in an osmotic pump tablet and administered orally for treatmentand prevention of restenosis.

What is claimed is:
 1. A compound of the formula ##STR42## wherein A andB are linkers which independently are C₁ -C₆ - alkylene, C₁ -C₆ -alkylenoxy, C₁ -C₆ - alkylenethio, or phenylene;X is NH, N--C₁ -C₆alkanoyl, O, or S; R₂ is hydrogen, (CH₂)_(n) Ph, where Ph is phenyl orphenyl substituted by one, two or three groups selected from halo, C₁-C₆ alkyl, C₁ -C₆ alkoxy, SH, C₁ -C₆ alkylthio, hydroxy, C₁ -C₆alkanoyl, --CN, --NO₂, C₁ -C₆ alkanoyloxy, --CF₃ or NR⁵ R⁶, and n is 0,1, 2, or 3,C₃ -C₆ -cycloalkyl, C₁ -C₆ alkanoyl, C₂ -C₆ alkenyl, and C₂-C₆ alkynyl, where the alkyl, alkenyl, and alkynyl groups may besubstituted by NR₅ R₆, phenyl, C₁ -C₆ alkylthio, C₁ -C₆ -alkyloxy,hydroxy, carboxy, halogen, C₃ -C₆ - cycloalkyl, or phenyl substituted byone, two or three groups selected from halo, C₁ -C₆ alkyl, C₁ -C₆alkoxy, SH, C₁ -C₆ alkylthio, hydroxy, C₁ -C₆ alkanoyl, --CN, --NO₂, C₁-C₆ alkanoyloxy, --CF₃ or NR⁵ R⁶, and where R₅ and ₆ are independentlyhydrogen, C₁ -C₆ alkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynyl, (CH₂)_(n) Phwhere Ph is phenyl or phenyl substituted by one, two or three groupsselected from halo, C₁ -C₆ alkyl, C₁ -C₆ alkoxy, SH, C₁ -C₆ alkylthio,hydroxy, C₁ -C₆ alkanoyl, --CN, --NO₂, C₁ -C₆ alkanoyloxy, --CF₃ or NR⁵R⁶, and n is 0, 1, 2, or 3, C₃ -C₆ - cycloalkyl, or R₅ and R₆ takentogether nitrogen to which they are attached can complete a ringselected from piperazine; Ar is phenyl, phenyl substituted by one, twoor three groups selected from halo, C₁ C₆ alkyl, C₁ C₆ alkoxy, SH, C₁ C₆alkylthio, hydroxy, C₁ C₆ alkanoyl, --CN, --NO₂, C₁ -C₆ alkanoyloxy,--CF₃ or NR₅ ₆, or the pharmaceutically acceptable salts thereof.
 2. Acompound of claim 1 having the formula ##STR43## wherein R₇ and R₈independently are C₁ -C₆ alkyl, C₁ -C₆ alkoxy, or halo.
 3. A compound ofclaim 2 wherein R₂ is C₁ -C₆ alkyl or C₁ -C₆ alkyl substituted bycarboxy.
 4. A compound of claim 3 wherein R₅ and R₆ independently are C₁-C₆ alkyl.
 5. A compound of claim 4 wherein A is C₁ -C₆ alkylenoxy.
 6. Acompound of claim 5 wherein B is phenylene.
 7. A compound of claim 6wherein X is NH.
 8. A compound of claim 2 having the formula ##STR44##wherein R₂ is C₁ -C₆ alkyl, A is C₁ -C₆ alkylenoxy, B is phenylene, andR₇ and R₈ are C₁ -C₆ alkyl, C₁ -C₆ alkoxy, or halo.
 9. The compoundwhich is6-(2,6-Dichlorophenyl)-2-4-diethylaminoethoxy)-phenylamino!-8-methyl-2-(pyridin-3-ylamino)-8H-pyrido2,3-d!pyrimidin-7-one-N.sup.ω -oxide; 6-(3,5-dimethoxyphenyl)-2- 4-2-diethylamino)ethoxy!-phenylamino!-8-ethyl-pyrido2,3-d!pyrimidin-7(8H)-one, N.sup.ω -oxide; 6-(3,5-dimethylthiophenyl)-2-4-(methylethylamino)phenyl!methyl!amino!-8-cyclopropyl-pyrido2,3-d!pyrimidin-7(8H)-thione, N.sup.ω -oxide;6-(3-methoxy-5-ethoxyphenyl)-2-2-(methylethylamino)ethyl!amino!-8-n-butyl-pyrido2,3-d!pyrimidin-7(8H)-one, N.sup.ω -oxide;6-(4-ethyl-5-methoxyphenyl)-2- 4-4-methyl-1-piperazinyl!butyl!amino!-8-methyl-pyrido2,3-d!-pyrimidin-7(8H)-one, N.sup.ω -oxide.
 10. A pharmaceuticalformulation comprising a compound of claim 1 in combination with apharmaceutically acceptable carrier, diluent, or excipient.
 11. A methodfor controlling proliferative disorders in a mammal selected from thegroup consisting of small-cell lung carcinoma, breast cancer, low gradehuman bladder carcinoma, human colorectal cancer psoriasis, and vascularsmooth muscle proliferation comprising administering to said mammal atherapeutically effective amount of a compound of claim
 1. 12. A methodfor treating small-cell lung carcinoma, breast cancer, low grade humanbladder carcinoma, human colorectal cancer comprising administering ananti-cancer amount of a compound of claim 1 to a mammal in need oftreatment.
 13. A method for treating atherosclerosis comprisingadministering an effective amount of a compound of claim 1 to a mammalin need of treatment.
 14. A method for treating psoriasis comprisingadministering to a mammal in need of treatment an anti-psoriatic amountof a compound of claim
 1. 15. A method for treating restenosiscomprising administering to a mammal in need of treatment an effectiveamount of a compound of claim 1.